Ebook Histotechnology - A self-Instructional text (3rd edition): Part 1

(BQ) Part 1 book Histotechnology - A self-Instructional text presents the following contents: Fixation, processing, instrumentation, safety, laboratory mathematics and solution preparation, nuclear and cytoplasmic staining, carbohydrates and amyloid, connective and muscle tissue.

Histotechnology A Self-Instructional Text 3rd Edition This page has been left intentionally blank Histotechnology A Self-Instructional Text 3rd Edition Freida L Carson PhD, HT(ASCP) Department of Pathology (retired) Baylor University Medical Center Dallas, Texas Christa Hladik AA, HT(ASCP)'m, QIHC Clinical Laboratory Manager, Neuropathology and Immunohistochemistry, UT Southwestern Clinical Laboratories, University of Texas Southwestern Medical Center Dallas, Texas • American Society for Clinical Pathology Press Publishing Team Adam Fanucci (Illustrations) Erik N Tanck & Tae W Moon (Design/Production) Joshua Weikersheimer (Publishing direction) Notice Trade names for equipment and supplies described are included as suggestions only In no way does their inclusion constitute an endorsement of preference by the Author or the ASCP The Author and ASCP urge all readers to read and follow all manufacturers' instructions and package insert warnings concerning the proper and safe use of products The American Society for Clinical Pathology, having exercised appropriate and reasonable effort to research material current as of publication date, does not assume any liability for any loss or damage caused by errors and omissions in this publication Readers must assume responsibility for complete and thorough research of any hazardous conditions they encounter, as this publication is not intended to be all-inclusive, and recommendations and regulations change over time Cover Images Image (left) : Hematoxylin eosin (H&E) - small intestine Image (middle): Papanicolaou- cervical smear Image (right): Aldan yellow-toluidine blue- gastric biopsy showing H pylori • American Society for Clinical Pathology Press Copyright© 2009 by the American Society for Clinical Pathology All rights reserved No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher Printed in Hong Kong 13 12 11 10 09 iv Table of Contents xvi Preface Chapter I Fixation 16 POTASSIUM DICHROMATE (K 2Cr20 ) 16 ZINC SALTS (ZnS0 4) 17 Other Fixative Ingredients 17 17 17 17 19 Definition 19 20 Functions of Fixatives 20 Actions of Fixatives Factors Affecting Fixation 20 20 20 TEMPERATURE SIZE VOLUME RATIO TIME 21 21 21 21 CHOICE OF FIXATIVE 21 PENETRATION 22 TISSUE STORAGE pH OSMOLALITY Reactions of the Cell with Fixatives THE NUCLEUS PROTEIN LIPIDS CARBOHYDRATES Simple Aqueous Fixatives or Fixative Ingredients ACETIC ACID 10 10 12 12 12 10 10 12 12 FORMALDEHYDE 12% Aqueous Formalin 12% Formalin Saline Calcium Formalin Formalin Ammonium Bromide Acetate Formalin 12% Neutralized Formalin 12% Neutral-Buffered Formalin Modified Millonig Formalin Alcoholic Formalin 13 GLUTARALDEHYDE 13 14 Phosphate-Buffered Glutaraldehyde GLYOXAL (C H 20 2) 14 MERCURIC CHLORIDE (HgCl 2) 15 OSMIUM TETROXIDE (OsO ) 15 PICRIC ACID Compound or Combined Fixatives B-5 FIXATIVE Stock Solution Working Solution Bouin Solution Gendre Solution Hollande Solution ZENKER AND HELLY (ZENKER-FORMOL) SOLUTIONS Zenker and Helly Stock Solution Zenker Working Solution Helly Working Solution Orth Solution Zamboni Solution (Buffered Picric Acid-Formaldehyde, or PAF) ZINC FORMALIN SOLUTIONS Aqueous Zinc Formalin (original formula) Unbuffered Aqueous Zinc Formalin Alcoholic Zinc Chloride Formalin 22 Nonaqueous Fixatives 22 ACETONE 22 ALCOHOL 22 23 23 23 23 23 23 25 Carnoy Solution Clarke Fluid Transport Solutions Michel Transport Medium PBS Buffer Stock Solution (also used in immunohistochemistry) PBS-10% Sucrose Solution Removal of Fixation Pigments Lugo) Iodine Solution 24 Troubleshooting Fixation Problems 24 AUTOLYSIS 24 INCOMPLETE FIXATION 27 References Histotechnology 3rd Edition v Chapter Processing Special Techniques in Processing 46 46 DECALCIFICATION 49 TROUBLESHOOTING DECALCIFICATION 49 FROZEN SECTIONS 50 TROUBLESHOOTING PROCESSING TISSUE FOR 33 Dehydration 33 ALCOHOLS 34 ACETONE 34 UNIVERSAL SOLVENTS 35 35 Clearing 35 TOLUENE 35 BENZENE 36 CHLOROFORM 54 Microscopes 36 ACETONE 54 LIGHT MICROSCOPE 36 ESSENTIAL OILS 55 POLARIZING MICROSCOPE 36 LIMONENE REAGENTS (XYLENE SUBSTITUTE) 55 PHASE-CONTRAST MICROSCOPE 36 ALIPHATIC HYDROCARBONS (XYLENE 55 DARKFIELD MICROSCOPE 56 FLUORESCENCE MICROSCOPE 56 ELECTRON MICROSCOPE XYLENE SUBSTITUTE) FROZEN SECTIONS 51 References Chapter Instrumentation 36 UNIVERSAL SOLVENTS 37 OTHER CLEARING AGENTS 57 Microtomes 37 Infiltration 57 ROTARY MICROTOME 37 PARAFFIN 57 SLIDING MICROTOME 40 WATER-SOLUBLE WAXES 57 CLINICAL FREEZING MICROTOME 40 CELLOIDIN 58 MICROTOME BLADES 41 PLASTICS 58 TROUBLESHOOTING MICROTOMY 41 AGAR AND GELATIN 30 41% SUCROSE 64 Cryostat 41 41 Troubleshooting Processing 65 65 Tissue Processors 66 MICROWAVE PROCESSOR 41 OVERDEHYDRATION 42 POOR PROCESSING 42 SPONGE ARTIFACT 67 67 AUTOMATIC STAINER 42 TISSUE ACCIDENTALLY DESICCATED 67 MICROWAVE STAINING OVEN 68 AUTOMATIC COVERSLIPPER PRECIPITATE IN THE PROCESSOR CHAMBER AND IN THE TUBING CONVENTIONAL PROCESSOR Stainers and Coverslippers 42 Embedding and Specimen Orientation 44 69 69 Miscellaneous Equipment Troubleshooting Embedding 44 SOFT MUSHY TISSUE 70 CHROMIUM POTASSIUM SULFATE-COATED 45 INCORRECT ORIENTATION 46 TISSUE CARRYOVER 70 POLY-L-LYSINE-COATED SLIDES 46 TISSUE NOT EMBEDDED AT THE SAME LEVEL 70 AMINOALKYLSILANE-TREATED SLIDES 46 PIECES OF TISSUE MISSING FROM THE BLOCK 71 DRYERS AND OVENS 72 CIRCULATING WATER BATH 72 FREEZERS AND REFRIGERATORS 72 pH METERS 74 BALANCES AND SCALES 74 EMBEDDING CENTER vi FLOTATION BATHS SLIDES 75 MICROMETER PIPETTES 75 SOLVENT RECYCLER 75 75 Instrument Quality Control 75 QUALITY CONTROL PROGRAM 16 ~~~~t:r s Laboratory Mathematics and Solution Preparation NEW INSTRUMENT VALIDATION 94 Percentage Solutions 95 Use of the Gravimetric Factor in Solution Preparation 96 Hydrates 96 Normal and Molar Solutions References ~~~J:!t~r ~ Safety 82 Biological or Infectious Hazards 82 TUBERCULOSIS EXPOSURE 82 CRYOGENIC SPRAYS 83 HIV, HEPATITIS C VIRUS (HCV), AND HBV 83 CREUTZFELDT-JAKOB DISEASE (CJD) 83 HANDLING TISSUE WASTE 83 Mechanical Hazards 84 ERGONOMICS 84 Chemical Hazards 86 PARTICULARLY HAZARDOUS SUBSTANCES (REPRODUCTIVE TOXINS, SELECT CARCINOGENS, AND SUBSTANCES WITH A HIGH DEGREE OF ACUTE TOXICITY) 86 CARCINOGENS 86 CORROSIVE SUBSTANCES 86 FIRE AND EXPLOSION HAZARDS 87 HAZARDOUS CHEMICAL SPILLS AND STORAGE 88 CHEMICAL STORAGE 88 HAZARDOUS CHEMICAL DISPOSAL 89 Hazard Identification General Safety Practices 90 90 EMPLOYEES 90 SUPERVISORS 91 References 97 The Metric System 97 TEMPERATURE CONVERSION 98 Buffers 98 General Guidelines for Solution Preparation, Use, and Storage 99 Stability of Solutions 99 References 101 ANSWERS TO PROBLEMS IN CHAPTER 101 ANSWERS TO PROBLEMS IN LEARNING ACTIVITIES ~~~~t:r ~ Nuclear and Cytoplasmic Staining 104 Ultrastructure of the Cell 104 THE NUCLEUS 105 THE CYTOPLASM 101 Staining Mechanisms 107 NUCLEAR STAINING 107 CYTOPLASMIC STAINING 108 The Dyes 109 FACTORS AFFECTING DYE BINDING 109 DIFFERENTIATION 109 THE NUCLEAR DYES 110 Harris Hematoxylin Delafield Hematoxylin Mayer Hematoxylin Ehrlich Hematoxylin 111 111 111 Histotechnology 3rd Edition vii 112 112 113 113 Gill Hematoxylin Scott Solution Weigert Hematoxylin Celestine Blue 114 PLASMA STAINS Eosin Counterstain Eosin-Phloxine B Counterstain 114 H&E Staining 114 MANUAL PROGRESSIVE STAINING METHOD 115 MANUAL REGRESSIVE STAINING METHOD 116 AUTOMATED STAINING 113 113 130 'troubleshooting Mounted Stained Sections 130 WATER BUBBLES NOTED IN MOUNTED SECTIONS 130 ALL AREAS OF SECTION CANNOT BE BROUGHT INTO FOCUS 130 CORN-FLAKING ARTIFACT SEEN ON MOUNTED 131 MOUNTED STAINED SECTIONS ARE NOT SECTIONS AS CRISP AS USUAL WHEN VIEWED MICROSCOPICALLY RETRACTED MOUNTING MEDIUM References 11 FROZEN SECTION STAINING 131 118 Troubleshooting the H&E Stain 132 118 INCOMPLETE DEPARAFFINIZATION 118 NUCLEAR STAINING IS NOT CRISP 11 PALE NUCLEAR STAINING 119 DARK NUCLEAR STAINING 120 RED OR RED-BROWN NUCLEI 120 PALE CYTOPLASMIC STAINING 136 Carbohydrates 120 DARK CYTOPLASMIC STAINING 136 GROUP 1: NEUTRAL POLYSACCHARIDES 120 EOSIN NOT PROPERLY DIFFERENTIATED 121 BLUE-BLACK PRECIPITATE ON TOP OF SECTIONS 121 HAZY OR MILKY WATER AND SLIDES 122 UNEVEN H&E STAINING 122 DARK BASOPHILIC STAINING OF NUCLEI AND CYTOPLASM, ESPECIALLY AROUND TISSUE Chapter Carbohydrates and Amyloid (NONIONIC HOMOGLYCANS) 136 GROUP II: ACID MUCOPOLYSACCHARIDES (ANIONIC HETEROGLYCANS) 136 GROUP III: GLYCOPROTEINS (MUCINS, MUCOID, MUCOPROTEIN, MUCOSUBSTANCES) 136 GROUP IV: GLYCOLIPIDS 137 Special Staining Techniques EDGES 122 POOR CONTRAST BETWEEN NUCLEUS AND CYTOPLASM 137 137 123 123 123 123 123 Nucleic Acid Stains FEULGEN REACTION Hydrochloric Acid, IN Schiff Reagent (De Tomasi Preparation) Sulfurous Acid 125 METHYL GREEN-PYRONIN Y Solution a-0.2M Acetic Acid Methyl Green-Pyronin Y Staining Solution 126 Polychromatic Stains 124 125 138 139 140 140 140 141 141 142 142 127 127 127 127 127 127 MAY-GRUNWALD GIEMSA STAIN Stock Jenner Solution Working Jenner Solution Stock Giemsa Solution Working Giemsa Solution Acetic Water, 1% 143 143 143 145 145 146 146 128 Mounting Stained Sections 128 RESINOUS MEDIA 129 AQUEOUS MOUNTING MEDIA 129 COVER SLIPS viii 146 146 147 147 147 PAS REACTION Periodic Acid, 0.5% Solution Schiff Reagent PAS REACTION WITH DIASTASE DIGESTION Periodic Acid, 0.5% Solution Potassium Metabisulfite, 0.55% Solution Phosphate Buffer, pH BEST CARMINE Carmine Stock Solution Working Carmine Solution MAYER MUCICARMINE Mucicarmine Stock Solution Mucicarmine Working Solution Weigert Iron Hematoxylin ALCIAN BLU E, PH 2.5 Acetic Acid, 3% Solution ALCIAN BLUE, PH 1.0 O.lN Hydrochloric Acid Solution 1% Alcian Blue Solution, pH 1.0 Nuclear-Fast Red Solution ALCIAN BLUE WITH H YALURONIDASE O.lM Potassium Phosphate, Monobasic Nuclear-Fast Red Solution 148 148 149 149 149 150 150 150 ALCIAN BLUE-PAS-HEMATOXYLIN 152 152 153 154 154 155 155 Amyloid 157 Acetic Acid, 3% Solution Aldan Blue, pH 2.5 Schiff Reagent MULLER-MOWRY COLLOIDAL IRON Ferric Chloride, 29% Solution Working Colloidal Iron Solution Nuclear-Fast Red Solution 172 173 173 173 174 174 175 175 176 177 ALKALINE CONGO RED METHOD Stock 80% Alcohol Saturated with Sodium Chloride CRYSTAL VIOLET Stock Saturated Crystal Violet Solution Iodine-Iodide Solution Crocein Scarlet-Acid Fuchsin Solution Phosphotungstic Acid, 5% Solution Alcoholic Safran Solution SILVER TECHNIQUES FOR RETICULAR FIBERS GOMORI STAIN FOR RETICULAR FIBERS Silver Nitrate, 10% Solution Potassium Permanganate, 0.5% Solution Sodium Thiosulfate, 2% Solution GORDON AND SWEETS STAIN FOR RETICULAR FIBERS 178 178 178 Silver Nitrate, 10% Solution Potassium Permanganate, 1% Solution Ferric Ammonium Sulfate, 2.5% Solution 179 179 MALLORY PTAH TECHNIQUE FOR THIOFLAVINE T FLUORESCENT METHOD Thioflavine T, 1% Solution References Chapter Connective and Muscle Tissue Staining Techniques for Muscle CROSS-STRIATIONS AND FIBRIN 180 180 180 180 181 181 PTAH Solution Gram Iodine Sodium Thiosulfate, 5% Solution Potassium Permanganate, 0.25% Solution PTAH WITHOUT MERCURIC SOLUTIONS Acidic Dichromate Solution 160 Connective 'tissue 182 Staining Technique for Basement Membranes 161 Basement Membrane 182 PERIODIC ACID-METHENAMINE SILVER MICROWAVE PROCEDURE FOR BASEMENT 161 Muscle 162 ~taining 162 163 164 165 165 166 166 167 168 168 168 168 170 170 171 171 171 MASSON TRICHROME STAIN 172 172 172 Techniques for Connective Tissue Fibers Bouin Solution Light Green Counterstain GOMORI 1-STEP TRICHROME STAIN Bouin Solution Acetic Acid, 0.5% Solution VAN GIESON PICRIC ACID-ACID FUCHSIN STAIN Acid Fuchsin, 1% Solution MEMBRANES 182 182 183 183 184 184 185 185 186 186 VERHOEFF ELASTIC STAIN 187 Lugo) Iodine Ferric Chloride, 10% Solution Sodium Thiosulfate, 5% Solution 187 ALDEHYDE FUCHSIN ELASTIC STAIN Aldehyde Fuchsin Solution Alcoholic Basic Fuchsin, 0.5% Solution Aldehyde Fuchsin Solution NOTES ON OTHER ELASTIC STAINS RUSSELL MODIFICATION OF THE MOVAT PENTACHROME STAIN Aldan Blue, 1% Solution Alkaline Alcohol Solution Stock Methenamine Silver Gold Chloride, 0.02% Solution Methenamine Silver Solution Gold Chloride, 0.2% solution Staining Techniques for Lipid OIL RED METHOD FOR NEUTRAL FATS Oil Red Stock Solution Oil Red Working Solution SUDAN BLACK B IN PROPYLENE GLYCOL Calcium-Formalin Solution OSMIUM TETROXIDE PARAFFIN PROCEDURE FOR FAT Osmium Tetroxide, 1% Solution 188 Staining Techniques for Connective 'tissue Cells 188 188 188 TOLUIDINE BLUE FOR MAST CELLS 190 References Toluidine Blue Solution METHYL GREEN-PYRONIN Y Histotechnology 3rd Edition ix • Fixative 10% neutral-buffered formalin is preferred Ferric Ammonium Sulfate, 2.5% Solution • Equipment Nonmetallic forceps, chemically clean glassware (Coplin jars, graduated cylinders, Erlenmeyer flasks, and pipettes), filter paper Ferric ammonium sulfate 2.5 g Distilled water lOOmL Formalin, 10% Solution • Technique Cut paraffin sections at to àm Quality Control Liver is a very good control tissue Formaldehyde, 37% to 40% lOmL Distilled water 90mL Gold Chloride, 0.2% Solution • Reagents Silver Nitrate, 10% Solution Silver n itrate 10 g Distilled water 100 mL Sodium Hydroxide, 3% Solution Sodium hydroxide 3g Distilled water 100 mL Ammoniacal Silver Solution Place mL of 10% silver nitrate solution in an Erlenmeyer flask, and add concentrated ammonium hydroxide, drop by drop, while shaking the container continuously, until the precipitate that forms is completely dissolved Do not add any excess ammonium hydroxide Add mL of 3% sodium hydroxide solution, and cautiously redissolve the precipitate with concentrated ammonium hydroxide until only a faint cloudiness remains If this step is carried too far and no cloudiness remains, add 10% silver nitrate solution, drop by drop, until drop causes the solution to become permanently cloudy Only a faint cloudiness is desirable Dilute the resulting solution to 50 mL with distilled water, and filter into a chemically clean Coplin jar Stock gold chloride solution (1 %) lOmL Distilled water 40mL Sodium Thiosulfate, 5% Solution Sodium thiosulfate 5g Distilled water lOOmL Nuclear-Fast Red (Kernechtrot) Solution Nuclear-fast red (Kernechtrot) 0.5 g Aluminum sulfate 25 g Distilled water 500mL Dissolve the aluminum sulfate in the distilled water, and then dissolve the nuclear-fast red in this solution using heat Cool, filter, and add a few grains of thymol as a preservative • Procedure Deparaffinize sections, and hydrate to distilled water Oxidize sections in 1% potassium permanganate solution for minutes Potassium Permanganate, 1% Solution Potassium permanganate 1g Rinse in tap water for minutes Distilled water lOOmL Bleach in 1% oxalic acid for minutes or until sections are colorless Wash in tap water for minutes Sensitize sections in 2.5% ferric ammonium sulfate for at least 15 minutes Wash in several changes of distilled water Oxalic Acid, 1% Solution Oxalic acid lg Distilled water lOOmL 178 Connective and Muscle Tissue I Ch I I [i8.20] A section of liver stained with the Gordon and Sweets technique No counterstain has been applied, the reticular network lining the sinusoids is well demonstrated, and the reticulin pattern can be evaluated easily with lower magnification Impregnate sections with the silver solution for minutes Rinse well with distilled water [i8.2 I] This is an example of a poor reticulin stain The reticulin pattern is totally obscured by the strong counterstaining The stain is unacceptable and should be repeated Silver stains are capricious, and when poor stains are obtained [i8.19], [i8.21] , the stain should be repeated All fresh reagents should be used, and particular attention should be paid to the preparation of the diamine silver solution 10 Reduce sections for minutes in the 10% formalin solution The methods presented not use uranyl nitrate, which has been banned in some laboratories because of its toxicity 11 Wash in tap water for minutes 12 Tone in 0.2% gold chloride solution for 10 minutes 13 Wash in tap water for minutes Staining Techniques for Muscle 14 Place slides in 5% sodium thiosulfate for minute MALLORY PTAH TECHNIQUE FOR CROSS-STRIATIONS 15 Rinse in distilled water AND FIBRIN 16 Counterstain, if desired, with nuclear-fast red for minutes 17 Wash well in distilled water 18 Dehydrate in changes each of 95% and absolute alcohols, clear in xylene, and mount with synthetic resin • Results [i8.20] • Reticulin [MALLORY 1942, CARSON 1984, SHEEHAN 1980] •Purpose The demonstration of muscle cross-striations and fibrin Crossstriations are a diagnostic feature of rhabdomyosarcomas or tumors arising from striated muscle Nemaline rods, present in some skeletal muscle diseases, may also be demonstrated by the method The PTAH has also been used for the demonstration of glial fibers and myelin This method is rarely used today because it has been replaced by immunohistochemical techniques Black • Other tissue elements depend on counterstain used • Technical Notes See the technical notes under the Gomori technique for reticular fibers The Gordon and Sweets method gives much less background and nuclear staining than most of the more common methods •Principle The amount of phosphotungstic acid in the staining solution is far greater than the amount of hematein (20:1), and it is believed that tungsten binds all available hematein to give a blue lake This metal-hematein lake stains selected tissue components blue, while the phosphotungstic acid is thought to stain the red-brown components This stain has been referred to as a polychrome stain because solution gives major colors The components colored red-brown will lose this color with water or prolonged alcohol washes, and therefore dehydration of the section after staining must be rapid Histotechnology 3rd Edition 179 • Fixative Zenker solution is preferred, but 10% neutral-buffered formalin may be used Potassium Permanganate, 0.25% Solution Potassium permanganate 0.25 g Distilled water 100 mL • Equipment Mechanical stirrer, Coplin jars, Erlenmeyer flasks, graduated cylinders Oxalic acid, 5% Solution • Technique Cut paraffin sections at to µm • Quality Control Use longitudinal sections of skeletal or cardiac muscle to demonstrate cross-striations, a section containing fibrin for the demonstration of fibrin • Reagents PTAH Solution Hematoxylin 1g Phosphotungstic acid 20 g Distilled water l,OOOmL Dissolve the solid ingredients in separate portions of water, dissolving the hematoxylin with the aid of heat When cool, combine No preservative is necessary The solution that is allowed to ripen naturally (approximately to months) is a better stain and lasts longer, but if time is not available for natural ripening, 0.2 g ofpotassium permanganate may be added to the solution This chemically ripened stain may be used immediately, but the best results are not obtained until the solution has been ripened for at least weeks or longer Oxalic acid Sg Distilled water lOOmL • Procedure Deparaffinize sections, and hydrate to distilled water If sections are formalin-fixed, mordant in Zenker fixative containing 5% acetic acid overnight at room temperature Rinse in tap water Place in Gram iodine for 15 minutes Rinse in tap water Place in 5% aqueous sodium thiosulfate for minutes Wash in tap water for 10 minutes Place sections in 0.25% potassium permanganate for minutes Rinse in tap water 10 Place in 5% oxalic acid for minute 11 Wash in running tap water for 10 minutes Gram Iodine 12 Stain in PTAH solution overnight at room temperature Iodine 3g Potassium iodide 6g Distilled water 900mL 13 Dehydrate rapidly through changes each of 95% and absolute alcohol, clear in xylene, and mount with synthetic resin Place the iodine, potassium iodide, and about 150 mL of the water in a flask, and stir until dissolved Add remaining water • Results [iB.22], [iB.23] • Cross-striations, fibrin Sodium Thiosulfate, 5% Solution Sodium thiosulfate Sg Distilled water lOOmL 180 Connective and Muscle Tissue I Ch Blue • Nuclei Blue • Collagen Red-brown • Technical Notes A chemically oxidized staining solution has a shorter shelf-life than a naturally ripened staining solution, because chemical oxidation may cause overoxidation When this occurs, there is a failure to show the proper density of the blue tones Solutions should be stored in amber glass bottles to retard overoxidation by light According to Meloan and Puchtler [1988], thorough washing of sections before staining is essential, because hydration of the tissue structures will greatly facilitate uptake of dye molecules They also state that sodium thiosulfate interferes with binding of the PTAH, so sections should be washed very well after application of sodium thiosulfate [iS.22] A section of Zenker-fixed skeletal muscle stained with the Mallory phosphotungstic acid-hematoxylin (PTAH) technique Bouin solution will also work as a mordant It should be used for hour at 60°C, and then the sections should be washed in running water until the yellow color is gone Skip the iodine step This gives better results than mordanting in Zenker solution or than the chromate method that follows [Wenk 2007] Fixation in zinc formalin may also provide satisfactory PTAH staining, but I have not tried this variant Fortunately, the PTAH technique has been largely replaced by immunohistochemical methods; although fibrin will stain very well after formaldehyde fixation, we consistently get better results when staining for muscle cross-striations if the tissue is mordanted, or postfixed, in a mercuric solution The stain is not as good when formalin -fixed sections are mordanted as when the original fixative is mercuric; very uneven staining frequently occurs in tissue originally fixed in formalin (iS.24] Stevens and Wilson [1996] described the following technique, which avoids mercuric solutions and uses acidic dichromate treatment instead PTAH WITHOUT MERCURIC SOLUTIONS [BANCROFT 1996] [iS.23] A very high magnification of a section of Zenker-fixed skeletal muscle stained with the Mallory PTAH technique.The A- and I-bands are well demonstrated, and the Z-lines can also be seen.The stain is rarely used today for the identification of cross-striation in tumors thought to be rhabdomyosarcomas; immunohistochemical staining is now preferred •Reagents Acidic Dichromate Solution HCl, 10% in absolute alcohol 12mL Potassium dichromate, 3% aqueous solution 36mL Acidic Potassium Permanganate Solution Potassium permanganate, 0.5% aqueous solution 50 mL Sulfuric acid, 3% solution 2.SmL Oxalic Acid, 1% Solution [iS.24] A section of formalin-fixed skeletal muscle stained with PTAH.The sections were mordanted overnight in Zenker fixative.This uneven staining appears to be characteristic of formalin-fixed tissue Note that the collagen is stained red in [i8.22], [i8.23], [i824] This is an important differentiation in correctly performed PTAH staining Oxalic acid lg Distilled water lOOmL Histotechnology 3rd Edition 181 •Procedure Deparaffinize sections, and hydrate to distilled water Place slides in acidic dichromate solution for 30 minutes Wash in tap water Place slides in acidic permanganate solution for minute Wash in tap water Bleach sections in 1% oxalic acid solution Rinse in' tap water Stain in PTAH solution overnight Dehydrate rapidly through 95% and absolute alcohols, clear in xylene, and mount with synthetic resin •Equipment Microwave oven or 50°C to 60°C water bath, vented plastic staining jars, graduated cylinders, Erlenmeyer flasks Technique Paraffin sections cut at àm Quality Control Kidney has an internal control No other control slide is necessary •Reagents The results of this stain should be the same as those described for the technique using mercuric solutions for either primary fixation or post-fixation mordanting • Technical Note Potassium dichromate is a hazardous chemical that is toxic on inhalation and ingestion It is corrosive to eyes, skin, and mucous membranes, and is also a carcinogen; however with care, the solutions pose little risk when used under normal conditions It cannot be put down the drain Staining Technique for Basement Membranes Stock Methenamine Silver Methenamine, 3% aqueous (15 g/500 mL) 400mL Silver nitrate, 5% aqueous (5 g/ 100 mL) 20mL Mix and keep solution refrigerated at 4°C Borax (Sodium Borate), 5% Solution Sodium borate 5g Distilled water lOOmL Working Methenamine Silver Solution Stock methenamine silver 25mL Distilled water 25mL Borax (sodium borate), 5% solution 2mL Prepare just before use PERIODIC ACID-METHENAMINE SILVER MICROWAVE PROCEDURE FOR BASEMENT MEMBRANES [BRINN 1983, SHEEHAN 1980] •Purpose This procedure best delineates basement membranes, and is most often used in the histopathology laboratory for the detection of abnormalities or diseases manifested in the glomerular basement membrane Periodic Acid, 1% Solution Periodic acid 1g Distilled water lOOmL Sodium Thiosulfate, 2% Solution •Principle The carbohydrate component of basement membranes is oxidized to aldehydes by periodic acid The aldehydes formed by oxidation bind the si lver ions from the methenamine silver complex and reduce the silver to its metallic form Methenamine gives the solution the alkaline properties necessary for the proper reaction, and the sodium borate acts as a buffer Toning is with gold chloride, and any unreduced silver is removed by sodium thiosulfate •Fixative 10% neutral-buffered formalin is preferred Mercury-containing fixatives are not recommended 182 Connective and Muscle Tissue I Ch Sodium thiosulfate 10 g Distilled water 500 mL Gold Chloride, 0.02% Solution Gold chloride, 1% solution mL Distilled water 49mL Stock Light Green, 0.2% Solution Light green SF (yellowish) 1g Distilled water SOOmL Glacial acetic acid mL Working Light Green Solution Light green stock solution lOmL Distilled water SOmL •Procedure This procedure is for slides If you not have slides, then include blank slides Do not use more than slides This procedure should be followed exactly for optimum results Deparaffinize sections, and hydrate to distilled water Place sections in 1% periodic acid solution for 15 minutes at room temperature Rinse in distilled water Place slides (5) in a vented plastic Coplin jar containing 50 mL of methenamine working solution.* Apply the vented cap, and place in the microwave oven Also place a vented plastic Coplin jar containing exactly 50 mL (measured) of distilled water in the oven Microwave on full power for exactly 70 seconds (see technical note 2) Remove both jars from the oven, mix the staining solution with a plastic Pasteur pipette, and let stand on the counter Check the slides frequently until the desire staining intensity is achieved This will take approximately 15 to 20 minutes [i8.25] A kidney glomerulus stai ned with a period ic acid-methenamine silve r technique A good stai n should look almost as if the basement membrane had been drawn with ink Although the tubular basement membranes are well-stai ned, they should not be used to judge the end point of t he impregnation; use the glomeru la r basement membrane on ly *If for some reason, the microwave oven cannot be used, substitute the following solutions and staining times: Methenamine Silver Solution Stock methenamine silver solution SOmL Borax, 5% solution SmL Preheat the solution, and stain slides at 56°C-60°C for 40-90 minutes Gold Chloride, 0.2% solution Gold chloride, 1% solution lOmL Distilled water 40mL Tone for 30 seconds to minute Rinse slides in the heated distilled water Tone sections in 0.02% gold chloride.* Rinse slides in distilled water Treat sections with 2% sodium thiosulfate for minute Wash in tap water 10 Counterstain in the working light green solution for I Yi minutes 11 Dehydrate with changes each of95% and absolute alcohols 12 Clear with xylene and mount with synthetic resin • Results [iS.25] • Basement membrane • Background Black Green • Technical Notes Sharper staining of the basement membrane and less background staining can be obtained with the use of the microwave oven for silver techniques The temperature is critical and should be just below boiling or approximately 95°C, immediately after removal from the microwave oven Each oven should be calibrated for the time required to reach the correct temperature Histotechn o logy 3rd Edition 183 Staining Techniques for Lipid Because the lipid stains requested in the routine histopathology laboratory are for simple lipids, only a selection of techniques for simple fats will be presented OIL RED METHOD FOR NEUTRAL FATS [i8.26] This kidney glomerulus has been understained with a periodic acid-methenamine silver technique Compare the staining here with that in [i8.25] Note that the basement membrane exhibits continuous staining in [i8.25] and interrupted staining here [PEARSE 1968] • Purpose The oil red method is used to demonstrate neutral lipids in frozen tissue sections Fat occurring in an abnormal place such as fatty emboli that may develop after either a bone fracture an injury that crushes a fatty body area may be demonstrated The fat stain may verify that the emboli caused death Degenerating material containing fat, such as cell membranes or myelin, may coalesce into fat droplets that are demonstrable with fat stains, and tumors arising from fat cells (liposarcomas) can be differentiated from other types of tumors • Principle Staining with oil-soluble dyes is based on the greater solubility of the dye in the lipoid substances than in the usual hydroalcoholic dye solvents This is a physical method of staining and the dye used must: be more soluble in the tissue lipid than in the solvent in which it is dissolved not be water soluble be strongly colored must act with tissue constituents only by solution [i8.27] Too much counterstain has been applied to th is section of kidney, and the period ic acid-methenamine silver staining is masked The section is also too thick; sections should be µm thick for demonstration of the glomerular basement membrane This is a very difficult stain to perform correctly Although the tubular membranes will stain, the end-point should be determined by the glomerular basement only When wellstained, the glomerular basement membrane should appear as a continuous black line Stopping the silver impregnation too soon will result in uneven or interrupted staining [i8.26] The application of too much counterstain will mask the silver stain and decrease contrast [i8.27] As with all silver stains, chemically cleaned glassware and plastic forceps should be used The PAS technique is also use to stain basement membranes and can be fo und in chapter 7, "Carbohydrates," pl37 The solvent used is critical, with isopropanol removing a minimal amount of lipid and propylene glycol not extracting any lipid • Fixative 10% neutral-buffered forma lin or calcium-formol Because of the lipid-dissolving ability, no alcoholic fixative shou ld be used • Equipment Cryostat, Coplin jars, Erlenmeyer flasks, graduated cylinders, filter paper • Technique Cut frozen sections at 10 µm Paraffin sections cannot be used because dehydrating and clearing agents dissolve the fat If freefloating sections are not used, then sections of fixed tissue should be picked up on coated, charged, or subbed slides • Quality Control Most tissue contains some fat, so normally a control is not used 184 Connective and Muscle Tissue I Ch •Reagents Oil Red Stock Solution Oil red 2.5 g lsopropanol, 98% 500 mL Mix well Oil Red Working Solution Oil red 0, stock solution 24mL Distilled water 16mL [i8.28] A frozen section from a fatty liver stained with oil red O.This tissue had been fixed in formalin for several weeks before freezing Note that even with the amount of fat present, the section shows little fat displacement Mix well, and let stand for 10 minutes Filter The filtrate can be used for several hours •Procedure Cut frozen sections, fix in 40% forma ldehyde for minute, and wash well in tap water Blot off excess water If the tissue has been previously fixed, there is no need to re fix Stain sections in oil red for 10 minutes Wash sections in tap water Stain for minute in Harris hematoxylin containing acetic acid (glacial acetic acid, mL/hematoxylin, 48 mL) Wash section in tap water Blue in ammonia water Wash in tap water Mount sections with an aqueous mounting medium Seal the edges of the coverslip with fingernai l polish [i8.29] Air bubbles were "chased out" by mashing on the coverslip with forceps during the mounting step Note the fat on top of this section compared with the duplicate cleanly mounted section in [i 8.28] ;fat is even present in the portal vein in this section Fat is relatively liquid, so the mounting step must be done carefully • Results [iB.28} • Fat Free-floating sections stain more readily; however, free-floating sections are difficult to obtain with the cryostat, and sections mounted on slides may be used The staining time may need to be adjusted Sections that have been fixed previously also may tend to loosen from the slides To improve the microtomy of frozen sections, forma lin-fixed tissues may be infiltrated with 30% sucrose solution before freezing Black According to method used Aqueous mounting media must be used, because the organ ic solvent present in synthetic resinous media will dissolve the fat • Technical Notes Lipids are removed by any fixative or solution containing alcohol or organic solvents, so sections cannot be processed for paraffin embedding Frozen sections are most frequently used, but the use of water-soluble wax for embedding will allow the demonstration of fat The fat in the section is relatively liquid and mobile, so care should be taken that no pressure is placed on the cover glass or the fat may be displaced [iS.29] If air bubbles are present in the section, remove the coverslip by soaking the slide in warm water If glycerin jelly is used for mounting, it should not be overheated, because this may melt the fat and also displace it • Other tissue elements Histotechnology 3rd Edition 185 Oil red may be dissolved in propylene glycol instead of isopropyl alcohol Prepare the solution and perform the stain as in steps though of the Sudan black B procedure that follows Steps through of the above procedure above should be used to complete the stain The oil red in propylene glycol staining should be done at 60°C Small fat droplets are less likely to be dissolved by propylene glycol than by alcohol SUDAN BLACK B IN PROPYLENE GLYCOL 1983] [CHIFFELLE 1951, CARSON • Purpose The demonstration of neutral lipids in tissue sections as described in the oil red procedure Sudan black B is the most sensitive of the lipid dyes and is far more soluble in phospholipids and, to a lesser extent in cerebrosides, than oil red It also is used in hematopathology to aid in differentiating granulocyte precursors from leukocytes committed to lymphocytic or monocytic pathways • Principle Sudan black B is one of the Sudan dyes used for demonstrating lipids In addition to the solubility of the dye in neutral fats as described with oil red 0, Sudan black B is a slightly basic dye and will combine with acidic groups in compound lipids; therefore, it will also stain phospholipids • Fixative 10% neutral-buffered formalin or sections postfixed in calciumformalin Because of lipid dissolving ability, no alcoholic fixative should be used • Equipment Cryostat, 100°C hot plate, 60°C oven, vacuum filtration apparatus with a fritted glass or Millipore filter, graduated cylinders, Erlenmeyer flasks, Coplin jars • Quality Control Most tissue contains some fat, so normally a control is not used Sudan Black B Staining Solution Sudan black B 0.7 g Propylene glycol lOOmL Add a small amount of the Sudan black Bat a time to th e propylene glycol, and stir continuously Heat to 100°C (do not exceed 110°C) for a few minutes, stirring constantly Filter immediately through Whatman No filter paper, cool, and refilter through a fritted glass filter or Millipore filter with a pore size suitable for filtering reagents This reagent may be stored indefinitely in a well-capped container in a 60°C oven • Procedure I Place fixed and rinsed cryostat sections in 100% propylene glycol for 15 minutes Use changes If tissue has not been previously fixed, fix the cut and mounted sections in calcium-formal for minute, rinse well, and then place in propylene glycol Stain sections in Sudan black B for 10 minutes Differentiate in 85% propylene glycol Wash sections well in distilled water Counterstain sections with nuclear-fast red for to minutes Wash well in several changes of distilled water Mount sections with an aqueous mounting medium • Results [i8.30] • Fat • Nuclei Blue-black Red • Technical Notes See the notes for oil red staining • Reagents Calcium-Formalin Solution Formaldehyde, 37% to 40% lOmL Calcium chloride 1.1 g Distilled water 90mL [i8.30] A frozen section from the same fatty liver shown in [iB.28] an d [iB.29] stained with Sudan black B 186 Connective and Muscle Tissue I Ch hours, depending on the denseness of the tissue: heart, hours; aorta, hour; and lung, hour This step should be done under the hood Periodically agitate the solution containing the tissue OSMIUM TETROXIDE PARAFFIN PROCEDURE FOR FAT [CARSON 1983] •Purpose The demonstration of fat by a method that allows paraffin embedding of the tissue Rinse tissue in changes of distilled water for 15 minutes each •Principle Differentiate by placing tissue in 0.5% periodic acid solution for 30 minutes Agitate the solution periodically The background tissue will clear and leave the fat stained black Wash the tissue in tap water for 30 minutes Process routinely, beginning with 70% alcohol; embed as usual 10% neutral-buffered formalin Cut paraffin sections to µm , pick up on slides, and dry routinely •Equipment Deparaffinize and hydrate sections as usual Osmium tetroxide chemically combines with fat, blackening it in the process This is the only method for fat that is chemical, although the nature of the black reaction product is not understood Fat that has combined with osmium tetroxide is insoluble in alcohols and xylene, and the tissue can be processed for paraffin embedding •Fixative Chemical hood, small capped bottles, processing cassettes • Technique Stain the wet tissue block, and then process in the tissue processor, beginning in 70% alcohol The gross section must be no thicker than mm, or the osmium will not penetrate 10 The sections may be stained with the routine H&E procedure or with any special stain desired Masson trichrome provides an excellent counterstain for osmium-fixed sections 11 After staining, the sections should be dehydrated, cleared, and mounted with a synthetic resin • Quality Control Most tissue contains some fat, so no control block is necessary •Reagents • Results [i8.31] • Fat Black • Other tissue elements According to method used Osmium Tetroxide, 1% Solution • Technical Notes Osmium tetroxide 1g Distilled water lOOmL Gross amounts of fat will not be fixed by this method, but small fat droplets and individual fat cells are beautifully demonstrated Store in a dark container in the refrigerator The vapor is harmful, so prepare and use the reagent under a fume hood Periodic Acid, 0.5% Solution Periodic acid 2.5 g Distilled water SOO mL •Procedure Trim 10% neutral buffered formalin-fixed tissue to mm thick, and wash in running tap water for at least 30 minutes Rinse the tissue well in distilled water Place the tissue in a small quantity (5 mL) of osmium tetroxide solution Cap the container, and leave for to [iS.31] This section of liver, containing a small amount of fat, was fixed with osmium tetroxide, and then processed and embedded in paraffin.The fat has been fixed and stained black by the osmium, so that it was not dissolved by the alcohol and xylene Following processing and microtomy, this section was stained with the Masson trichrome procedure Histotechnology 3rd Edition 187 The block should be faced carefully and sections should be taken as soon as possible Osmium has a very low penetrating power and the interior of the block may not show good fat preservation and staining It is essential that the sections for processing be cut thin The cytoplasm will be gray as a result of the osmium, and this will affect the quality of the cytoplasmic stains Staining Techniques for Connective Tissue Cells TOLUIDINE BLUE FOR MAST CELLS [LILLIE 1965, VACCA 1985] [i8.32] A section of skin from a mastocytoma in a dog Note the many metachromatically stained mast cells in the section [Image courtesy of Lott R, •Purpose The demonstration of mast cells in tissue Mast cells play a key role in inflammation, and allergic reactions, are implicated in the pathology associated with autoimmune disorders, and are found in mast cell tumors (mastocytomas) common in dogs and cats and rare in humans •Principle Mast cells will stain metachromatically with toluidine blue; that is, they will stain a different color from the dye solution and the rest of the tissue The control section should show mast cells stained red-purple (metachromatic staining) and the background stained blue (orthochromatic staining) The color shift, called metachromasia, generally is attributed to the cationic or basic dye and is somewhat dependent on pH, dye concentration, and temperature Blue or violet dyes will show a red color shift, and red dyes will show a yellow color shift with metachromatic tissue elements Birmingham, AL] •Procedure Deparaffinize sections, and hydrate to distilled water Stain sections in toluidine blue solution for 10 minutes Rinse in distilled water Quickly dehydrate with 95% and absolute alcohol Clear in xylene, and mount with synthetic resin • Results [i8.32] • Mast cells • Background •Fixative 10% neutral-buffered formalin is preferred • Equipment Coplin jars, Erlenmeyer flask, graduated cylinder Deep rose-violet Blue • Technical Notes Alcohol cannot be used for dehydration in most metachromatic staining procedures, but metachromasia of mast cell granules is stable and will not be lost after alcoholic dehydration A rapid screening method for mast cells is to simply apply a light methylene blue stain as if you were counterstaining an acid-fast stain The mast cells are very well demonstrated (i8.33] • Technique Cut paraffin sections at to µm Mast cells will also be stained orange-red by the methyl greenpyronin stain as shown in [i8.34] • Quality Control A section containing mast cells must be used as a control METHYL GREEN-PYRONIN Y This method was described in detail in chapter 6, pl24, as a method of differentiating DNA and RNA It is mentioned again in this chapter because it stains plasma cells, which are connective tissue cells Because of the rough endoplasmic reticulum present, with its high RNA content, the cytoplasm of mature plasma cells and immunoblasts will be stained pink to red by pyronin (i8.35] •Reagents Toluidine Blue Solution Toluidine blue 0.1 g Distilled water 100 mL 188 Connective and Muscle Tissue I Ch [i8.33] mast cells can be seen in this section, which was stained with the methylene blue counterstain used in the Kinyoun acid-fast technique This solution must be applied so that a very light stain is achieved, but it provides a rapid, easily performed screening method for mast cells [i8.35] Several mature plasma cells of this lymph node stained with the methyl green-pyronin stain show dark rose-stained cytoplasm (RNA) The larger immunoblasts have more and paler rose-stained cytoplasm The nuclei (DNA) are stained blue-green and the nucleoli (RNA), prominent in the immunoblasts, are also stained rose [i8.34] This mast cell tumor of the skin has been stained with the methyl green-pyronin stain Mast cell granules are very pyroninophilic, but the color is more orange than that seen with RNA staining Histotechnology 3rd Ed ition 189 References Bancroft JO, Stevens A [1996] Theory and practice of histological techniques New York, NY: Churchill Livingstone Brinn NT [1983] Rapid metallic histological staining using the microwave oven./ Histotechnol 6:125 Carson FL, Pickett JP [1983] Histochemistry In: Race GJ, ed Laboratory Medicine, Philadelphia, PA: Harper & Row Carson FL, Pickett JP, Matthews JL [1984] Preparation of tissue for laboratory examination In: Race GJ, ed Laboratory Medicine, Philadelphia, PA: Harper & Row Chiffelle TL, Putt FA [1951] Propylene and ethylene glycol as solvents for Sudan IV and Sudan black B Stain Technol 26:51 Churukian CJ [1993] Manual of the Special Stains Laboratory of the Department of Pathology and Laboratory Medicine 6th ed Rochester, NY: University of Rochester Medical Center Crowder CH [1991] Microwave and Conventional Procedures: A Manual of Histologic Techniques Louisiana State University, Baton Rouge, LA Gomori G [1950a] Aldehyde fuchsin: a new stain for elastic tissue Am l Clin Pathol 20:665 Gomori G [1950b] A rapid one-step trichrome stain Am l Clin Pathol 20:661 Gordon H, Sweets HH [1936] A simple method for the silver impregnation of reticulum Am J Pathol 12:545 Kiernan JA [1990] Histological & Histochemical Methods: Theory & Practice, 2nd ed New York, NY: Pergamon Press Lillie RD, Fullmer HM [1976] Histopathologic Technic and Practical Histochemistry New York, NY: McGraw-Hill Book Co Luna LG [1968] Histologic Staining Methods of the Armed Forces Institute of Pathology 3rd ed New York, NY; McGrawHill Book Co Luna LG [1992] Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts Gaithersburg, MD: American Histolabs Inc, Publications Division Mallory FB [1942] Pathologic Technic Philadelphia, PA: WB Sanders Meloan SN, Puchtler H [1988] On the chemistry of phosphotungstic acid-hematein: development of a rapidly ripening PTAH solution J Histotechnol 11: 153 Mowry RW [1979] Aldehyde fuchsin staining, direct or after oxidation: problems and remedies, with special reference to human pancreatic B cells, pituitaries and elastic fibers Stain Technol 53: 141 Pearse AGE [1968] Histochemistry 3rd ed Baltimore, MD: Williams & Wilkins Co Sheehan DC, Hrapchak BB [1980] Theory and Practice of Histotechnology, 2nd ed Columbus, OH: Battelle Press Stevens A, Wilson I [1996] The haematoxylins and eosin In: Bancroft JD, Stevens A, eds Theory and Practice of Histological Techniques New York, NY: Churchill Livingstone Sweat F, Meloan SN, Puchtler H [1968] A modified one-step trichrome stain for demonstration of fine connective tissue fibers Stain Technol 43:227 Vacca LL [1985] Laboratory Manual of Histochemistry New York, NY: Raven Press Wenk P [1995] Personal communication Lillie RD [1965] Histopathologic Technic and Practical Histochemistry 3rd ed Philadelphia, PA: Blakiston 190 Connective and Muscle Tissue I Ch Wenk P [2007] Personal communication LEARNING ACTIVITIES I Perform the Masson trichrome, Gomori trichrome, van Gieson, Verhoeff, aldehyde fuchsin, Russell modification of the Movat pentachrome, silver impregnation for reticulum, Mallory PTAH, oil red 0, Sudan black B, toluidine blue, methyl green-pyronin, and methenamine-silver staining procedures You may choose any tissue that will demonstrate positive staining (Note: the van Gieson technique may be used as a counterstain with the Verhoeff stain and not done as a separate stain.) Microscopically examine each stained section, and compare the results with those given in the procedure If the results are unsatisfactory, analyze the procedural steps for possible sources of error If a mistake is identified, repeat the stain after correcting the problem, and reexamine the slides Histotechnology 3r d Edition 191 This is supposed to be page 192 The publisher left it blank ... GREEN-PYRONIN Y Solution a- 0.2M Acetic Acid Methyl Green-Pyronin Y Staining Solution 12 6 Polychromatic Stains 12 4 12 5 13 8 13 9 14 0 14 0 14 0 14 1 14 1 14 2 14 2 12 7 12 7 12 7 12 7 12 7 12 7 MAY-GRUNWALD GIEMSA... YALURONIDASE O.lM Potassium Phosphate, Monobasic Nuclear-Fast Red Solution 14 8 14 8 14 9 14 9 14 9 15 0 15 0 15 0 ALCIAN BLUE-PAS-HEMATOXYLIN 15 2 15 2 15 3 15 4 15 4 15 5 15 5 Amyloid 15 7 Acetic Acid, 3%... BINDING 10 9 DIFFERENTIATION 10 9 THE NUCLEAR DYES 11 0 Harris Hematoxylin Delafield Hematoxylin Mayer Hematoxylin Ehrlich Hematoxylin 11 1 11 1 11 1 Histotechnology 3rd Edition vii 11 2 11 2 11 3 11 3 Gill