odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb

NSC 17525 No of Pages 11 January 2017 Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 Neuroscience xxx (2017) xxx–xxx ODORANT RECEPTOR PROTEINS IN THE MOUSE MAIN OLFACTORY EPITHELIUM AND OLFACTORY BULB VICTORIA F LOW AND PETER MOMBAERTS * Max Planck Research Unit for Neurogenetics, Max-von-Laue-Strasse 4, D-60438 Frankfurt, Germany Abstract—In the mouse, odorant receptor proteins (ORs) are G-protein-coupled receptors expressed in mature olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE) ORs mediate odorant reception at the level of the OSN cilia Most $1100 OR genes in the mouse genome are expressed, at the RNA level, in mature OSNs The literature on antibodies against ORs is limited, and most reports are with antibodies that are not commercially available Here we have screened 40 commercial antibodies against human and mouse ORs by immunofluorescence staining of coronal cryosections of the MOE of 21-day-old C57BL/6J mice Various methods of antigen retrieval were tested Of the 19 antibodies raised against human ORs, three yielded a consistent immunoreactive signal in the mouse MOE; of these three, two appeared to cross react against one or more, unknown, mouse ORs Of the 21 antibodies raised against mouse ORs, six yielded a consistent immunoreactive signal in the mouse MOE; of these six, two also stained specific glomeruli in the olfactory bulb Antibody specificity could be validated with gene-targeted mouse strains in the case of three ORs The number of OSNs immunoreactive for the MOR28/Olfr1507 antibody is greater in C57BL/6J than in 129S6/SvEvTac wild-type mice Taken together, our results are encouraging: 20–30% of these commercially available antibodies are informative in immunohistochemical analyses of the mouse MOE The commercial availability of these antibodies should facilitate the study of OR proteins in the MOE and the olfactory bulb, and the replicability of results in the literature Ó 2016 The Author(s) Published by Elsevier Ltd on behalf of IBRO This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/) Key words: olfactory receptor, olfactory sensory neuron, glomerulus, antibody, immunofluorescence, antigen retrieval *Corresponding author E-mail addresses: vicky.low@gen.mpg.de (V F Low), peter.mombaerts@gen.mpg.de (P Mombaerts) Abbreviations: aa, amino acid; AR, antigen retrieval; EDTA, ethylenediaminetetraacetic acid; GFP, green fluorescent protein; IRES, internal ribosome entry site; MOE, main olfactory epithelium; OMP, olfactory marker protein; OR, odorant receptor; OSN, olfactory sensory neuron; PBS, phosphate-buffered saline; PFA, paraformaldehyde; SDS, sodium dodecyl sulfate INTRODUCTION In mammals such as mouse and rat, odorants are detected in the main olfactory epithelium (MOE) by olfactory sensory neurons (OSNs) via odorant receptors (ORs), which are G-protein-coupled seventransmembrane proteins (Buck and Axel, 1991) In the mouse, there are 1099 OR genes with an intact open reading frame, of which 1,087 are expressed at the RNA level in OSNs (Saraiva et al., 2015) Due to the difficulty in raising antibodies against G-protein-coupled receptors, there have been relatively few reports about OR proteins in the literature Initial studies with antibodies against rat ORs suggested that the antigen is localized to the cilia of OSNs in the rat MOE (Koshimoto et al., 1992; Krieger et al., 1994) In situ hybridization against OR RNAs in mouse revealed that OR gene expression is restricted to OSNs within discrete regions of the MOE called zones (Ressler et al., 1993; Miyamichi et al., 2005) Gene-targeted mice enabled the visualization of entire OSNs expressing a given OR by targeted integration of a cassette that affords cotranslation of the OR with a marker such as taulacZ via an internal ribosome entry site (IRES) (Mombaerts et al., 1996) Axons of OSNs expressing the same OR coalesce into two or a few glomeruli per mouse olfactory bulb, which reside in highly restricted regions (Ressler et al., 1994; Mombaerts et al., 1996; Zapiec and Mombaerts, 2015) Replacement of the OR coding region showed that the OR does not only mediate odorant detection (Bozza et al., 2002), but is also a determinant of where in the olfactory bulb the axons coalesce and form glomeruli (Mombaerts et al., 1996; Feinstein et al., 2004; Feinstein and Mombaerts, 2004) The widespread success of the gene-targeted approach of the OR-IRES-marker design may have overshadowed or diminished efforts to raise and characterize OR antibodies But these gene-targeted mutations are designed to visualize the OSNs (including their cilia, dendrite, cell body, axon, and axon terminals) that express a given OR, and not enable the visualization of the subcellular localization of the OR protein within the OSNs An attempt at creating an OR: GFP C-terminal fusion protein by gene targeting resulted, for reasons that remain unclear, in altered positions of the corresponding glomeruli in the olfactory bulb (Feinstein et al., 2004) Antibodies against mouse ORs revealed that OR proteins are present not only in OSN cilia, dendrite, and cell body but also within the axon and axon terminals 10 http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 0306-4522/Ó 2016 The Author(s) Published by Elsevier Ltd on behalf of IBRO This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 NSC 17525 No of Pages 11 January 2017 V F Low, P Mombaerts / Neuroscience xxx (2017) xxx–xxx 87 (Barnea et al., 2004; Strotmann et al., 2004), thus supporting the notion that the OR protein is involved in axonal wiring of OSNs A quarter of a century after the discovery of mammalian OR genes (Buck and Axel, 1991), there are still few papers on immunofluorescence staining of the mouse MOE and olfactory bulb with OR antibodies that are commercially available There are numerous antibodies against ORs on the market, but they remain largely uncharacterized The signal yielded by such antibodies can be affected by the high sequence homology between ORs, by post-translational modifications, by protein folding, by protein–protein interactions, and by fixation crosslinking To increase the accessibility of the antigen to the antibody, antigen retrieval can be used to break some of the crosslinks formed during fixation and to alter the protein re-folding following heat denaturation (Shi et al., 2001; Emoto, 2005) Epitope unmasking through antigen retrieval can allow for a more accurate detection, and therefore representation, of the antigen Here, we describe our screening of 40 commercially available antibodies against human and mouse ORs in the mouse MOE using various antigen retrieval techniques, in coronal 12-lm sections of the MOE of 21- day-old C57BL/6J mice We obtained reliable immunoreactive signals with two antibodies against human ORs (due to presumed crossreactivity with mouse ORs) and six antibodies against mouse ORs in the mouse MOE Of these six, two antibodies also stained specific glomeruli in the olfactory bulb 88 EXPERIMENTAL PROCEDURES 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 89 Mice 90 Mice were 21 days old Wild-type mice were C57BL/6J or 129S6/SvEvTac Our gene-targeted strains (Feinstein et al., 2004) are publicly available from The Jackson Laboratory (Bar Harbor, ME, USA): mI7-IRES-tauGFP (https:// www.jax.org/strain/006664), and M50-IRES-GFP-IREStaulacZ (https://www.jax.org/strain/006686) The genetargeted strain MOR28-IRES-gap-GFP (Serizawa et al., 2000) is publicly available from the RIKEN BioResource Center (Tsukuba, Japan) (https://www2.brc.riken.jp/lab/animal/detail.php?brc_no=RBRC02928) Mice were maintained in specified pathogen-free conditions in individually ventilated cages of the Tecniplast green line Mice received ad libitum gamma-irradiated ssniff V1124-727 (ssniff, Soest, Germany) Nesting, bedding, and enrichment were provided as nestpak, Datesand Grade (Datesand, Manchester, United Kingdom) Mouse experiments were performed in accordance with the German Animal Welfare Act, European Communities Council Directive 2010/63/EU, and institutional ethical and animal welfare guidelines of the Max Planck Research Unit for Neurogenetics All efforts were made to minimize the number of animals used and their suffering 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 Immunohistochemical staining 113 Tissue preparation Mice were anesthetized by injection of ketamine HCl and xylazine (120 mg/kg and 114 mg/kg body weight, respectively) and perfused with ml ice-cold phosphate-buffered saline (PBS), followed by 15 ml 2% paraformaldehyde in PBS (PFA) The mouse heads were dissected, postfixed in 2% PFA for h at °C, and decalcified in 0.45 M EDTA in PBS overnight at °C Samples were cryoprotected in, successively, 15% and 30% sucrose in PBS at °C, frozen in O.C.T Compound (Tissue-Tek), sectioned at 12 lm with a Leica CM3500 cryostat, and collected onto glass slides Alternative fixation methods were also tested; perfusion with 4% PFA resulted in poorer antigenicity, and immersion fixation in 2% PFA for h without perfusion did not yield improved antigenicity 115 Antigen retrieval Various antigen retrieval steps were tested to optimize visualization of the target protein These included a 10-min incubation in M HCl at room temperature, or 10 in ice-cold 100% methanol followed by 0.5% sodium dodecyl sulfate (SDS) in PBS at room temperature Antigen retrieval with a pressure cooker (2100 Retriever; BioVendor) was carried out with 10 mM citric acid pH 6.0 and 10 mM Tris pH 9.0 with or without 0.05% Tween 20 and mM EDTA After heating in a pressure cooker to 121 °C, slides were left to cool for h in the retrieval buffer Additionally, PBS washes were tested with 0.05% Tween 20 or 0.2% Triton X-100, and incubation with antibody was tested for various times during a period of 1–4 days, with and without 0.1% Triton X-100 Best results were obtained with PBS washes and incubation with antibody diluted with 0.1% Triton X-100 There was no obvious difference in antigen detection with different incubation times 128 Immunohistochemistry Slides were washed  10 with PBS, and, if necessary, antigen retrieval was carried out and the slides were again washed Slides were then blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories) or normal donkey serum (Millipore) in PBS for h at room temperature After blocking, slides were incubated with the primary antibodies in 1% normal goat serum or normal donkey serum with 0.1% Triton X-100 in PBS overnight at °C Commercial rabbit antibodies against ORs were tested at dilutions of 1:200, 1:500, and 1:2000 The following antibodies against human ORs were obtained from Osenses (Keswick, Australia): OR11H4, OR1B8, OR1D4, OR1E1, OR1L8, OR1M1, OR1N2, OR2A4, OR2D3, OR2F1, OR2H1, OR2K2, OR2T1, OR2V1, OR2W1, OR4C11, OR51E1, OR8A1 The following antibodies against mouse ORs were obtained from Osenses: Olfr2, Olfr6 (x2; different peptide sequences), Olfr15, Olfr16, Olfr24, Olfr56, Olfr151, Olfr156, Olfr362, Olfr412, Olfr521, Olfr545, Olfr552, Olfr554, Olfr685, Olfr749, Olfr831, Olfr1507 We also tested sheep OR51E1 and chicken Olfr73 antibodies (Osenses), and rabbit Olfr1303 antibody (Life Technologies) Mature OSNs were visualized with goat OMP antibodies (olfactory marker protein; 1:1000; Wako Chemicals), and green fluorescent protein (GFP) was detected with chicken GFP antibodies (1:1000; Aves Labs) After incubation with primary antibodies, slides 146 Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 116 117 118 119 120 121 122 123 124 125 126 127 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 NSC 17525 No of Pages 11 January 2017 V F Low, P Mombaerts / Neuroscience xxx (2017) xxx–xxx 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 were washed with PBS, then incubated with secondary antibodies: donkey anti-goat Alexa488, donkey antisheep Alexa488, goat anti-chicken Alexa488, donkey anti-rabbit Alexa555, goat anti-rabbit Alexa594 (1:1000; Invitrogen), and DAPI (1:10,000; Molecular Probes) for h at room temperature Slides were washed with PBS and coverslipped with Mowiol (Calbiochem) Combined immunohistochemistry and in situ hybridization Tissue preparation Mice were anesthetized by injection of ketamine HCl and xylazine (120 mg/kg and mg/kg body weight, respectively) and perfused with 15 ml ice-cold 4% PFA The mouse heads were dissected, postfixed in 4% PFA, and decalcified in 0.45 M EDTA in PBS overnight at °C Samples were cryoprotected in 15% and 30% sucrose in PBS at °C, frozen in O.C.T Compound (Tissue-Tek), sectioned at 12 lm with a Leica CM3500 cryostat, and collected onto glass slides In situ hybridization and immunohistochemistry OR RNAs were detected with biotinylated riboprobes against Olfr156 (Strotmann et al., 1999); Olfr412 (forward 50 -ATGGACGGAGGCAACCAGA-30 , reverse 50 -GGCTT CCTTAGCAGTCTTCCC-30 ), and Olfr552 (Addgene plasmid #15843; Hirota et al., 2007) Riboprobes were generated as described in Ishii et al (2004) Combined immunohistochemistry and in situ hybridization was carried out using a modified protocol from Ishii et al (2004) Briefly, slides were washed with PBS and antigen retrieval was carried out with 10 mM citric acid pH 6.0 Next the slides were fixed with 4% PFA for 15 min, washed with PBS, followed by blocking endogenous peroxidases with 0.1% H2O2 in PBS for 30 Slides were washed in PBS, incubated in 0.2 M HCl for 10 min, followed by another PBS wash Slides were then incubated for 10 in 0.1 M Triethanol amine-HCl, 0.25% acetic anhydrate; pH 8.0 After being washed in PBS, slides were dehydrated in graduated ethanol and left to air-dry Probes were diluted in hybridization solution and the slides were incubated overnight at 65 °C Slides were then washed as described in Ishii et al (2004), blocked with TNB (0.5% NEN Blocking reagent in TN; 100 mM Tris–Cl pH 7.5, 150 mM NaCl) for h at room temperature, and incubated with streptavidin–HRP (1:1500; PerkinElmer) and an OR antibody: rabbit Olfr156 antibody, rabbit Olfr412 antibody, and rabbit Olfr552 antibody (1:2000; Osenses) diluted in TNB with 0.1% Triton X-100, for nights at °C Slides were then washed thoroughly with TN-T (TN with 0.05% Tween 20) Streptavidin-labeled biotinylated probes were visualized by incubation with tyramide-fluorescein (1:50; PerkinElmer) diluted with 1 amplification diluent (PerkinElmer) for 10 Finally, in order to visualize the antibody, slides were washed with TN-T and incubated with goat anti-rabbit Alexa594 (1:1000; Invitrogen) and DAPI (1:10,000) for h at room temperature Slides were washed with TN before coverslipping with Mowiol (Calbiochem) 215 Image analysis 232 Microscopy Following labeling with immunohistochemistry, and immunohistochemistry combined with in situ hybridization, slides were examined under a Zeiss LSM 710 confocal microscope 233 Cell counting Every fifth section from the mouse MOE was labeled with rabbit Olfr1507 antibody (visualized with Alexa 594) and chicken GFP antibody (visualized with Alexa 488) Slides were scanned with a Pannoramic Midi Scanner (3D Histech) The diameter of cell nuclei was measured with Panoramic Viewer Software (3D Histech) Each section was exported as a tiff file using the Panoramic Viewer Software, and cells were counted using the cell counter plugin in Fiji ImageJ version 1.50 g 237 Statistics Statistical analysis was performed using GraphPad Prism Values are shown as the mean ± standard error of the mean A t-test was used to compare between two means For multiple comparisons, the one-way ANOVA Newman–Keuls multiple comparison test was used to test for significance Significance was set at P 0.05 246 Table List of antibodies against ORs Antigen Host Antigen peptide region Catalogue Source Optimal antigen retrieval Optimal antibody dilution OR1D4 OR1L8 OR51E1 Rabbit Rabbit Rabbit Not specified aa 200–250 aa 270–317 OSR00252W OSR00258W OSR00201W Osenses Osenses Osenses Citric acid AR Citric acid AR Citric acid AR 1:2000 1:2000 1:2000 Olfr2 Olfr6 Olfr156 Olfr412 Olfr552 Olfr1507 Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit aa 270–325 aa 275–316 Not specified aa 260–312 aa 270–317 aa 265–313 OSR00025G OSO00046W OSR00044W OSR00211W OSR00204W OSR00212W Osenses Osenses Osenses Osenses Osenses Osenses Citric acid AR Citric acid AR, Tris AR Citric acid AR, methanol/SDS Citric acid AR No AR, Citric acid AR No AR, Citric acid AR 1:2000 1:2000 1:2000 1:2000 1:2000 1:5000 List of commercial antibodies raised against human and mouse ORs that yielded immunostaining in cryosections of the mouse MOE As the peptide sequence of the antigens is not available, the region is given from which the antigenic peptide was designed Antigen retrieval conditions, and optimal dilutions of the antibodies are indicated aa; amino acid, Citric acid AR; Antigen retrieval with citric acid buffer pH 6.0 in a pressure cooker, No AR; No antigen retrieval, Tris AR; Antigen retrieval with Tris buffer pH 9.0 in a pressure cooker Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 234 235 236 238 239 240 241 242 243 244 245 247 248 249 250 251 252 NSC 17525 No of Pages 11 January 2017 V F Low, P Mombaerts / Neuroscience xxx (2017) xxx–xxx 253 RESULTS 254 We screened 40 commercially available antibodies that were raised against human or mouse ORs in the mouse MOE, testing various antigen retrieval methods to optimize visualization of the antigen Of these 40 antibodies, 19 were raised against human ORs with $80% amino acid identity to mouse ORs, and 21 were against mouse ORs Here we present the results from of the antibodies that yielded reproducible immunostaining in the mouse MOE (Table 1) For the sake of brevity, we refer to the antibody with the name of the OR against which it was raised: ORxxx for human ORs, and Olfrxxx for mouse ORs 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 Olfr2, one of the Olfr6 antibodies (OSO00046W), Olfr156, Olfr412, Olfr552, and Olfr1507 The Olfr2 and Olfr6 antibody labeling was observed in the ventral-lateral aspect of the MOE, and appeared weak (Fig 2) Olfr2 labeling was best observed after antigen retrieval in the pressure cooker with citric acid, and Olfr6 labeling was observed with both citric acid and Tris antigen retrieval buffers in the pressure cooker Low signal could be due to low protein expression of the Olfr2 and Olfr6 ORs, low sensitivity of the antibodies, or suboptimal fixation and antigen retrieval procedures Fortunately, there are gene-targeted mouse strains available for these two ORs, respectively mI7-IREStauGFP (Bozza et al., 2002) and M50-IRES-GFP-IRES- Screening of antibodies against human ORs in the mouse MOE Of the 19 antibodies against human ORs, three yielded consistent immunoreactivity when antigen retrieval was carried out in a pressure cooker with citric acid: OR1D4, OR1L8, and OR51E1 Both the OR1D4 antibody (Fig 1A, B0 ) and the OR1L8 antibody (Fig 1C, D0 ) colabeled with the OMP antibody, suggesting that they cross react with one or more mouse OR proteins in the MOE The mouse ORs with the closest homology to these human ORs are Olfr412 (81% identity with OR1D4 across the entire amino acid sequence of the OR) and Olfr355 (78% identity with OR1L8 in the amino acid region 200–250 from which the antigenic peptide was designed) For the OR1D4 and OR1L8 antibodies, immunoreactivity was observed midway along the dorsal-medial and ventral-lateral aspect of the MOE OR51E1 immunoreactivity was observed more ventral-laterally The rabbit OR51E1 antibody reacted with an antigen in the mouse MOE (OR51E1 has 87% identity with mouse Olfr558 in the amino acid region 270–317 from which the antigenic peptide was designed): signal was detected in cell bodies, but there was no evidence of immunoreactivity in cilia or dendrites Furthermore OR51E1 immunoreactivity did not colocalize with OMP immunoreactivity (Fig 1E, F0 ) Screening of antibodies against mouse ORs in the mouse MOE Of the 21 antibodies against mouse ORs, we observed consistent immunoreactivity with six antibodies: Fig Labeling of the mouse MOE by antibodies against human ORs (A) OR1D4-immunoreactive cells in the MOE (red) Mature OSNs were detected with OMP antibody (green) Merge with DAPI (blue) (B) High magnification of insert in A showing weak OR1D4 antibody labeling in the dendrite and cell body of OMP-immunoreactive cells in the MOE (merged red and green, B0 ) (C) OR1L8immunoreactive cells labeled in the MOE (red) Mature OSNs were detected with OMP antibody (green) Merge with DAPI (blue) (D) High magnification of insert in C Antibody labeling is observed in the cilia and cell body, and colocalized with OMP-immunoreactive cells (merged red and green, D0 ) (E) OR51E1 labeling was detected in a population of cells in the MOE (red) Mature OSNs were detected with OMP antibody (green) Merge with DAPI (blue).(F) High magnification of insert in E demonstrating strong OR51E1 immunoreactivity in the cell bodies of a population of cells that were not OMP-immunoreactive (merged red and green, F0 ) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 310 311 312 313 314 315 316 317 318 319 320 321 322 323 NSC 17525 No of Pages 11 January 2017 V F Low, P Mombaerts / Neuroscience xxx (2017) xxx–xxx Fig Labeling of the mouse MOE by antibodies against mouse ORs Olfr2 and Olfr6 (A) Olfr2-immunoreactive cells (red) in the MOE of an mI7IRES-tauGFPÀ/À mouse These cells were also detected with GFP antibody (green) Merge with DAPI (blue) (B) High magnification of insert in A showing Olfr2 immunoreactivity in the cilia, dendrite, and cell body, and colocalization with GFP antibody signal (merged red and green, B0 ) (C) Olfr2 immunoreactivity was not detected in a GFP-immunoreactive (green) glomerulus of an mI7-IRES-tauGFPÀ/À mouse Merge with DAPI (blue) (C0 ) (D) Olfr6 immunoreactivity (red) was weak, and only occasionally detected in association with GFP immunoreactivity (green) in an M50-IRESGFP-IRES-taulacZÀ/À mouse Merge with DAPI (blue) (E) High magnification of insert in D Weak Olfr6 immunoreactivity is observed in the cell body, dendrite and cilia of cells that express M50-IRES-GFP-IRES-taulacZ (merged red and green, E0 ) (F) Olfr6 immunoreactivity was not detected in GFP-immunoreactive (green) glomeruli of an M50-IRES-GFP-IRES-taulacZÀ/À mouse Merge with DAPI (F0 ) Scale bar = 20 lm (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 taulacZ (Feinstein et al., 2004) These strains enabled us to validate that Olfr2 (Fig 2A, B0 ) and Olfr6 (Fig 2D, E0 ) antibody labeling was limited to cells that were also GFP positive, indicating that the antibody is specific to the respective OR Neither antibody against Olfr2 (Fig 2C, C0 ) nor Olfr6 (Fig 2D, D0 ) detected protein expression in the GFP-positive glomeruli in the olfactory bulb The Olfr156 antibody yielded signal midway along the dorsal-medial and ventral-lateral aspect of the MOE, and appeared to have low immunoreactivity (Fig 3A, B0 ) SDS/methanol antigen retrieval sporadically reveled positive labeling, and citric acid antigen retrieval yielded more reproducible signal In both cases only cilia were labeled consistently, and the cell body, and dots within the dendrites, were visible on rare occasions It is possible that the antigen is more accessible in certain subcellular localizations; there may be modifications occurring to the OR protein that allow detection under certain conditions; or an increased density of OR protein in certain subcellular localizations, such as in the cilia, may make it more easy to visualize The Olfr412 antibody showed strong immunoreactivity following citric acid antigen retrieval, and signal was detected in the dorsal-medial aspect of the mouse MOE (Fig 3C, D0 ) This pattern differs from the OR1D4 antibody, which yielded signal midway along the dorsal-medial and ventral-lateral aspect of the MOE Thus, although Olfr412 is the mouse OR with the highest homology (80%) to human OR1D4, it is unlikely to be the antigen that the OR1D4 antibody detects The Olfr552 antibody also showed strong immunoreactivity in the mouse MOE (Fig 3E, F0 ) This antibody did not require antigen retrieval, but the signal was greatly improved following antigen retrieval with citric acid Signal was detected through the dorsalmedial aspect of the mouse MOE Two glomeruli were detected in the olfactory bulb Consistent with Olfr552 belonging to class I ORs, the labeled glomeruli resided within the class I dorsal domain of the olfactory bulb The medial glomerulus was located midway along the anterior-posterior extent, and the lateral glomerulus (Fig 3G) was located more anteriorly For Olfr156, Olfr412, and Olfr552, there are no genetargeted mice available to validate that the antibody is specific for the respective OR Instead we used in situ hybridization combined with immunohistochemistry to verify that these antibodies were labeling the intended OR Combining these methods compromises the quality of both techniques, and we were unable to detect antibody labeling with Olfr156 in combination with in situ hybridization We were able to detect both protein and RNA in the mouse MOE for Olfr412 (Fig 4A, B0 ) and Olfr552 (Fig 4C, D0 ) We could thus verify that Olfr412 Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 NSC 17525 No of Pages 11 January 2017 V F Low, P Mombaerts / Neuroscience xxx (2017) xxx–xxx Fig Labeling of the mouse MOE by antibodies against mouse ORs Olfr156, Olfr412, and Olfr552 (A) Olfr156-immunoreactive cells (red) Mature OSNs were detected with the OMP antibody (green) Merge with DAPI (blue) (B) High magnification of insert in A showing Olfr156 immunoreactivity in the cilia and dendrite Merge with OMP immunoreactivity (B0 ) (C) Olfr412immunoreactive cells (red) Mature OSNs were detected with OMP (green) Merge with DAPI (blue) (D) High magnification of insert in C showing Olfr412 immunoreactivity in the cilia, dendrite and cell body, colocalizing with OMP immunoreactivity (merged, D0 ) (E) Olfr552-immunoreactive cells (red) Mature OSNs were detected with OMP (green), and merged with DAPI (blue) (F) High magnification of insert in E showing strong Olfr552 immunoreactivity in the cilia, dendrite and cell body of cells that are labeled with OMP antibody (F0 ) (G) Olfr552 immunoreactivity was also detected in a glomerulus Merge with DAPI Scale bar = 20 lm (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 378 379 380 381 382 383 384 385 386 387 388 389 390 391 and Olfr552 antibody labeling was closely associated with the signal from respective riboprobes in the mouse MOE, indicating the antibodies are labeling the cells that express the respective OR gene The sensitivity and specificity of these antibodies could not be verified using this method of combined labeling Finally, the Olfr1507 antibody showed strong immunoreactivity in the ventral-lateral aspect of the mouse MOE (Fig 5), and did not require antigen retrieval to be visualized Signal could be detected in the cilia, dendrite, and cell body of OSNs Immunostaining colocalized with GFP expression in mice homozygous for the MOR28-IRES-gap-GFP gene-targeted mutation (Serizawa et al., 2000) (Fig 5A, B0 ) We also detected strong immunoreactivity in GFPpositive glomeruli in the olfactory bulb (Fig 5C, C0 ) Having a reliable antibody that detects a mouse OR for which we have a gene-targeted mouse strain available gave us the opportunity to investigate monoallelic expression of Olfr1507 in mice heterozygous for MOR28-IRES-gapGFP, with the other, wild-type MOR28/Olfr1507 allele either of C57BL/6J origin (abbreviated B6) or 129S6/SvEvTac origin (abbreviated 129) (Fig 5D, E0 ) The MOR28/ Olfr1507 gene targeting had been carried out in an embryonic stem cell line of 129 origin (Serizawa et al., 2000) We observed an even distribution of cells that are doubly GFP positive and antibody positive throughout the MOE, versus cells that are antibody-only positive In the olfactory bulb we found that the GFP-positive glomeruli were labeled with the Olfr1507 antibody in MOR28-IRES-gap-GFP+/À mice (Fig 5F, F0 ) The glomeruli did not appear to be homogeneously labeled with Olfr1507 and GFP antibodies in MOR28-IRES-gap-GFP+/À mice Instead, there is a segregation of axon terminals expressing the genetargeted and wild-type Olfr1507 alleles This segregation was observed in MOR28-IRES-gap-GFP+/À (129) mice (Fig 5F’) and in MOR28-IRESgap-GFP+/À (B6) mice (data not shown) 392 Counting Olfr1507-immunoreactive cells in the MOE 430 We counted Olfr1507-immunoreactive cells in every fifth coronal section of the MOE from five types of mice, 20 in total: MOR28-IRES-g ap-GFPÀ/À (n = 5), MOR28-IRESgap-GFP+/À (B6) (n = 4), MOR28IRES-gap-GFP+/À (129) (n = 3), wild-type C57BL/6J (n = 5), and wild-type 129S6/ SvEvTac (n = 3) mice To correct for overcounting in sections, we performed an Abercrombie correction (Abercrombie, 1946; Bressel et al., 2016) (Table 2) We observed no significant difference in the nuclear diameter between strains, and used a correction factor of 0.68–0.69 In all five types of mice, we observed a distinct ‘M’-pattern distribution of Olfr1507-immunoreactive OSNs in the MOE, with the peaks corresponding to the ectoturbinates of the MOE (Fig 6A) The first Olfr1507immunoreactive cells were observed midway along the anterior–posterior dimension, between 3.5 and mm Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 NSC 17525 No of Pages 11 January 2017 V F Low, P Mombaerts / Neuroscience xxx (2017) xxx–xxx 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 (B6) and MOR28-IRES-gap-GFP +/À (129) mice, we observed a homogeneous distribution of the two populations throughout the MOE (Fig 6A) However, there was a slight preference for expression of the wild-type, B6-derived allele in MOR28-IRES-gap-GFP+/À (B6) mice, with 56.38 ± 1.9% of cells counted expressing the wild-type Olfr1507 allele and 43.01 ± 1.8% of cells expressing the mutant Olfr1507 allele (P < 0.002; Fig 6B) (An additional 0.62 ± 0.4% of the cells counted reacted with the GFP antibody only.) In MOR28-IRESgap-GFP (129) mice, we found that 51.12 ± 0.8% of cells expressed the wild-type, 129-derived allele and 47.21 ± 0.1% of cells expressed the gene-targeted allele (P < 0.008) (An additional 1.67 ± 0.8% of cells counted reacted with the GFP antibody only.) Although the expression of the 129 wild-type Fig In situ hybridization combined with immunohistochemistry in the mouse MOE (A) The vs gene-targeted allele is still same sells were detected with the Olfr412 antibody (red) and the Olfr412 riboprobe (green) Merge statistically different, it is closer to with DAPI (blue) (B) High magnification of insert in A showing Olfr412 immunoreactivity in the cilia, dendrite, and cell body, and colocalizing with Olfr412 riboprobe hybridization in the cell body (B0 ) the expected 50/50 distribution than (C) Cells were detected with Olfr552 antibody (red) and Olfr552 riboprobe (green) Merge with DAPI when the wild-type allele is of B6 (blue) (D) High magnification of insert in C showing Olfr552 immunoreactivity in the cilia, dendrite, origin Note that expression of the and cell body, and colocalizing with Olfr552 riboprobe hybridization detected in the cell body (D ) gap-GFP reporter is driven by a Scale bar = 20 lm (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 129-derived promoter sequence in the gene-targeted mutation (Serizawa et al., 2000) from the start of the MOE The expression peaked after Further support for the idea that approximately mm, then began to drop off, but the probability of expression of Olfr1507 is higher for the expression quickly increased again, and continued to B6 allele than for the 129 allele, came from comparing the most posterior portion of the MOE the numbers of immunoreactive cells in the various In MOR28-IRES-gap-GFPÀ/À mice, we found that strains In MOR28-IRES-gap-GFPÀ/À mice we counted 0.94 ± 0.4% of the counted cells were labeled with only 31,657 ± 2305 Olfr1507-immunoreactive cells, and the Olfr1507 antibody but not with the GFP antibody, these numbers were not statistically different from suggesting that the antibody is highly specific for MOR28-IRES-gap-GFP+/À (129) mice (36,897 ± Olfr1507-expressing cells in the mouse MOE 1547), or 129S6/SvEvTac wild-type mice (24,985 ± Conversely, in these mice only 0.40 ± 0.2% of the 1448) (Fig 6C) Likewise, the numbers of cells counted counted cells were labeled with the GFP antibody but in MOR28-IRES-gap-GFP+/À (B6) mice (41,988 ± not with the Olfr1507 antibody, indicating that the 3142) and in C57BL/6 wild-type mice (43,500 ± 3606) antibody is also highly sensitive Therefore, we conclude were not significantly different from each other, but were that the antibody against an Olfr1507 peptide that is significantly higher than in MOR28-IRES-gap-GFPÀ/À commercially available from Osenses (OSR00212W) is mice and in 129S6/SvEvTac wild-type mice (one-way a reliable, specific, and sensitive detector of Olfr1507ANOVA P < 0.003; Fig 6C) Incidentally, we did not expressing OSNs in the mouse MOE, with 98.66 ± identify a significant correlation between the number of 0.4% of counted cells labeled with both the Olfr1507 Olfr1507-immunoreactive OSNs counted in the MOE antibody and the GFP antibody in MOR28-IRES-gap-GF and the weight of the mice, indicating that the PÀ/À mice (Fig 6B) differences in cell numbers were not due to trivial Next, we examined the two populations of Olfr1507variations in mouse weight (Fig 6D) expressing OSNs for each allele in MOR28-IRES-gapGFP+/À mice OSNs that were labeled with both the DISCUSSION Olfr1507 and GFP antibodies were considered to express the MOR28-IRES-gap-GFP gene-targeted Here, we have demonstrated that some commercially allele, and OSNs that were labeled with only the available antibodies against ORs can be a reliable tool Olfr1507 antibody were considered to express the to examine cells expressing a given OR in the mouse wild-type allele In both MOR28-IRES-gap-GFP+/À MOE Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 NSC 17525 No of Pages 11 January 2017 V F Low, P Mombaerts / Neuroscience xxx (2017) xxx–xxx Fig Labeling of the MOE of MOR28-IRES-gap-GFPÀ/À and +/À mice by antibodies against Olfr1507/MOR28 (A) Olfr1507-immunoreactive cells (red) in a MOR28-IRES-gap-GFPÀ/À mouse are colabeled with GFP antibody (green) Merge with DAPI (blue) (B) High magnification of insert in A Olfr1507 immunoreactivity is detected in the cell body, dendrite, and cilia, and colocalizes with GFP immunoreactivity (B0 ) (C) Olfr1507 immunoreactivity colocalizes uniformly with GFP immunoreactivity within a glomerulus of a MOR28-IRES-gap-GFPÀ/À mouse Merge with DAPI (C’) (D) Olfr1507-immunoreactive cells (red) in a MOR28-IRES-gap-GFP+/À (129) mouse are colabeled with GFP antibody (green) Merged with DAPI (blue) (E) High magnification of insert in D Olfr1507 immunoreactivity is detected in the cell body, dendrite, and cilia, and colocalizes with GFP immunoreactivity (E’) (F) Olfr1507 immunoreactivity (red) segregates within a GFP-immunoreactive (green) glomerulus of an MOR28-IRESgap-GFP+/À (129) mouse Merge with DAPI (F0 ) Scale bar = 20 lm (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table Abercrombie correction; Nucleus diameter of OSNs Strain MOR28-IRES-gap-GFPÀ/À MOR28-IRES-gap-GFP+/À (B6) MOR28-IRES-gap-GFP+/À (129) C57BL/6J 129S6/SvEvTac Nucleus diameter (lm) Abercrombie correction factor 5.60 ± 0.13 5.46 ± 0.11 5.43 ± 0.10 0.68 0.69 0.69 5.40 ± 0.12 5.30 ± 0.09 0.69 0.69 The average nucleus diameter and the standard error of the mean and the calculated Abercrombie factors are given for the five types of mice Scale bars = 20 lm 540 Antibodies against ORs 541 We have demonstrated that a substantial fraction (at least 20%) of commercially available antibodies are a viable way for assessing OR expression in the mouse MOE We found that tissue fixation had an effect on the quality of antigen detection, with perfusion with 2% PFA being optimal For the antibodies that we were unable to get to work, other fixation methods may yield better and consistent signals We also tested various antigen retrieval methods, and found that when antigen retrieval 542 543 544 545 546 547 548 549 was required, the best results were often obtained with antigen retrieval in a pressure cooker with a citric acid buffer, pH 6.0 While alternative antigen retrieval methods also worked, we found that if signal could still not be detected after antigen retrieval with citric acid, alternative methods would also be without success Some antibodies yielded strong signals, and for others the signal was barely or inconsistently detectable It would be interesting to determine if the variation in signal is a result of variable antigenicity of the antibodies, or reflects widely differing levels of protein expression within OSNs expressing these particular ORs We confirm and extend the observations of OR immunoreactivity in the cell body and dendrite as well as in the cilia of OSNs (Barnea et al., 2004; Strotmann et al., 2004) In the dendrite, and sometimes in the cell body of OSNs, a distinct dot-like pattern could be detected This pattern of signal could be an artifact of antibody staining Nevertheless, it is a repeatable observation with various antibodies against ORs, and we reported it earlier in the M71::GFP fusion mouse strain (Feinstein et al., 2004), suggesting that this dot-like pattern of OR protein signal reflects a biological process It could be related to the visualization of specific forms of protein folding, it may be due to increased density of protein as a result of pulsed transcription, or it may be related to protein trafficking This issue can be examined further Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 NSC 17525 No of Pages 11 January 2017 V F Low, P Mombaerts / Neuroscience xxx (2017) xxx–xxx Fig Numbers of Olfr1507-immunoreactive cells in the MOE of wild-type and gene-targeted mice (A) Average number of cells counted per coronal section through the anterior–posterior dimension of the MOE in five types of mice: MOR28-IRES-gap-GFPÀ/À, MOR28-IRES-gap-GFP+/ À (B6), MOR28-IRES-gap-GFP+/À (129), C57BL/6J, and 129S6/SvEvTac Cells labeled with the Olfr1507 antibody (red), the GFP antibody (green), and both the Olfr1507 and GFP antibodies (orange) (B) Percentages of cells labeled with the Olfr1507 antibody (red), the GFP antibody (green), and both the Olfr1507 and GFP antibodies (orange) in three types of mice: MOR28-IRES-gap-GFPÀ/À, MOR28-IRES-gap-GFP+/À (B6), and MOR28-IRES-gap-GFP+/À (129) mice (C) Comparison of the number of cells counted in each strain Cell counts in every fifth section were multiplied by five, and subjected to Abercrombie correction (D) There is no correlation between the numbers of Olfr1507-immunoreactive cells and mouse weight (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 577 578 579 580 581 through the use of antibodies against ORs in combination with membrane markers, and with super-resolution microscopy In situ hybridization combined with immunohistochemistry 593 Due to the high homology of mouse ORs, a gene-targeted mouse strain is ideal to validate specificity and to assess sensitivity of an OR antibody We have demonstrated that in some cases immunohistochemistry can be combined with in situ hybridization as a form of validation, but this technique has its limitations It can indicate that an antibody is targeting the population of ORs that is intended, but until this method is further improved, it cannot reveal how specific or sensitive an antibody is Correlating OR RNA expression with the corresponding OR protein expression could reveal interesting information about OR transcription and translation within OSNs 594 Glomerular labeling 595 In most cases we were unable to detect a reliable signal in glomeruli But with 3,600 glomeruli in the mouse olfactory bulb (Richard et al., 2010), it remains possible that we overlooked the glomerular signal for some antibodies For two antibodies (Olfr552 and Olfr1507/MOR28) we were able to visualize specific glomeruli Interestingly, both of these antibodies did not require antigen retrieval to be detected Even when antigen retrieval was performed, the glomeruli were still detected, suggesting that antigen retrieval itself was not inhibiting the detection of glomeruli with the other antibodies 582 583 584 585 586 587 588 589 590 591 592 596 597 598 599 600 601 602 603 604 605 In both MOR28-IRES-gap-GFP+/À (B6) and MOR28-IRES-gap-GFP+/À (129) mice, we observed a compartmentalization of immunoreactive signal within the glomeruli: they were labeled uniformly with the Olfr1507 antibody, but visualized only partially with the GFP antibody The segregation in glomeruli in MOR28IRES-gap-GFP+/À (B6) mice has been reported previously (Ishii et al., 2001), and there is a single amino acid polymorphism between B6 (isoleucine) and 129 (valine) at residue 50 Interestingly, we also observed a compartmentalization of the two populations of OSNs in glomeruli of MOR28-IRES-gap-GFP+/À (129) mice This compartmentalization may reflect a subtle difference in RNA or protein expression level between the wild-type and 129 alleles, or an undocumented effect of the gapGFP axonal marker In this marker, the 20 N-terminal amino acid residues of GAP43 are fused to the N-terminus of the GFP to target it to the plasma membrane (Moriyoshi et al., 1996) 606 Probability of OR gene expression 625 We used MOR28-IRES-gap-GFPÀ/À mice (Serizawa et al., 2000) as validation that the Olfr1507 antibody is highly specific and sensitive for Olfr1507-expressing OSNs We also determined that Olfr1507-expressing OSNs are distributed in an ‘M’-like pattern in the MOE in a series of coronal sections We have described a similar pattern of distribution of OSNs in the MOE for OSNs expressing mI7/Olfr2, M50/Olfr6, or SR1/Olfr124 (Bressel et al., 2016) The number of Olfr1507expressing OSNs in a 21-day-old C57BL/6J mouse 626 Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 627 628 629 630 631 632 633 634 635 NSC 17525 No of Pages 11 January 2017 10 V F Low, P Mombaerts / Neuroscience xxx (2017) xxx–xxx 666 (43,500 ± 3606) is slightly higher than the number of MOR256-17-expressing OSNs (37,023 ± 6318) (Bressel et al., 2016) When we assessed Olfr1507 expression in MOR28IRES-gap-GFP+/À (B6) mice, we found that there was a preferential expression of the wild-type, B6-derived allele We observed a reduced preference of expression of the wild-type, 129-derived allele in MOR28-IRES-gapGFP+/À (129) mice When we compared the number of Olfr1507-expressing OSNs in wild-type mice, we found that there were more cells in B6 mice than in 129 mice, suggesting that the probability of Olfr1507 gene choice is higher when driven by the B6 promoter than the 129 promoter There does appear to be an additional effect of the targeted mutations in terms of a slight reduction in the probability of gene choice Interestingly, Olfr1507 is the most proximal OR gene among a cluster of seven OR genes that are regulated by the H element (Tsuboi et al., 1999; Serizawa et al., 2000, 2003) When the H element is deleted by gene targeting, the expression of Olfr1507 is abolished entirely, and the expression of the more distal OR genes is reduced in a distance-correlated fashion (Fuss et al., 2007; Nishizumi et al., 2007; Khan et al., 2011) Moreover, the position of the Olfr1507 coding region in C57BL/6 mice is 11 kb closer to the H element compared to mice with a 129 background (Fuss et al., 2007) We speculate that the difference in distance of the H element to the Olfr1507 gene is causally related to the difference in probability of gene choice, measured operationally as the number of Olfr1507-expressing OSNs 667 CONCLUSIONS 668 Antibodies against mouse ORs are a valuable tool for understanding the biological distribution of OR proteins, and to characterize OR-specific glomeruli in the olfactory bulb when no gene-targeted strain is available Their value to olfactory neuroscience may have been overshadowed by the success of the gene-targeted approach The significance of reports based on custom-designed or home-made polyclonal antibodies is constrained by the lack of the commercial or public availability of these reagents, hampering replicability studies Here we have characterized 40 commercially available antibodies against ORs We have demonstrated that they have a reasonable probability of success for immunofluorescence in the MOE (20–30%), and can be highly specific and sensitive The production of monoclonal antibodies against ORs will be an 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olfactory epithelium and olfactory bulb Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 772 773 774 775 776 777 778 779 ... H (2005) Continuous and overlapping expression domains of odorant receptor genes in the olfactory epithelium determine the dorsal/ventral positioning of glomeruli in the olfactory bulb J Neurosci... This issue can be examined further Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb Neuroscience (2017),... the anterior–posterior dimension, between 3.5 and mm Please cite this article in press as: Low VF, Mombaerts P Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb