A Guide to Fixation and Preservation of Tissues

CHAPTER 1: INTRODUCTION TO TISSUE STORAGE 
Background 
Tissues 
Epithelial Tissue 
Connective Tissue 
Muscle Tissue 
Nerve Tissue 
Organs 
CHAPTER 2: TISSUE PRESERVATION 
Introduction 
Tissue Fixation and Fixatives 
Classification, Action, and Applications 
Fixative Agents 
Aldehydes 
Formaldehyde 
Glutaraldehyde 
Other Aldehydes 
Other Cross-Reacting Fixatives 
Oxidizing Agents 
Osmium Tetroxide 
Chromic Acid Protein-denaturing Agents 
Methyl and Ethyl Alcohol 
Acetic Acid 
Fixatives with Unknown Mechanisms 
Picric Acid 
Mercuric Chloride 
Acetone 
Fixation Mixtures 
Additives for Fixation 
Tannic acid 
Phenol 
Heavy Metal Solutions 
Lanthanum 
Lithium 
Detergents 
Microwave (MW) Irradiation 
Microwave Fixation of Brain 
MW to Accelerate Fixation 
Other Fixation Strategies 
Excluded Volume Fixation 
Vapor Fixation 
Freeze-drying 
Post-fixation Treatment 
Summary 
CHAPTER 3: PROCEDURAL FACTORS INVOLVED IN FIXATION 
Temperature 
Size of Specimens and Penetration of Fixative 
Changes in Volume 
pH and Buffers 
Osmolarity 
Concentration of Fixatives 
Duration of Fixation 
Fixation of Specific Substances 
Glycogen 
Lipids 
Proteins 
Mucoids 
Nucleic Acids and Nucleoproteins 
Biogenic Amines 
Enzymes 
Fixation Artifacts 
CHAPTER 4: FIXATION PROTOCOLS FOR SPECIFIC SUBSTANCES 
Glycogen 
Lipids 
Elftman’s fixative 
Reagents Required 
Method 
Swank and Davenport’s Fixative 
Reagents Required 
Method 
Mucoids 
Lillie’s Alcoholic Lead Nitrate 
Reagents Required 
Method 
Cetylpyridinium chloride (C.P.C.) 
Reagents Required 
Method 
Lead Subacetate-ethanol Fixative 
Reagents Required 
Method 
Biogenic Amines 
Potassium Dichromate-acetate-formaldehyde Fixative 
Reagents Required 
Method 
Recovery of DNA, RNA, and Protein with 70% Ethanol Fixation 
Technique 
Low-melt Polyester Embedding 
Method A: Large Tissue Specimens (several cm size) 
Method B: Small Tissue Specimens and Biopsies (mm to 2 cm size) 
Histology Slide Cutting Procedure 
Paraffin Embedding 
DNA, RNA, and Protein Analysis 
Tissue Slide Preparation from Laser Capture Microdissection (LCM) 
Slide Preparation from Manual Microdissection Sample 
PCR Amplification of DNA 
Formalin-fixed, Paraffin-embedded Tissue 
Summation 
CHAPTER 5: CRYOPRESERVATION OF TISSUE 
Hypothermic Storage 
Vitrification 
The Cryopreservation Process 
Cryopreservation Case Study 
Ovarian Tissue 
Related Processes 
Freeze-Drying 
Supercooling 
Tissue Storage: Equipments and Supplies 
CHAPTER 6: SUGGESTED READING 
APPENDIX FORMULATIONS FOR VARIOUS FIXATIVES 
Formaldehyde Solutions 
10% Neutral Buffer Formalin (4% formaldehyde) 
Reagents Required 
Baker’s Formol-calcium (modified) 
Reagents Required 
Formol Saline 
Reagents Required 
Alcoholic Formaldehyde 
Reagents Required 
Paraformaldehyde 
Reagents Required 
Method 
Buffered Formaldehyde-glutaraldehyde 200 mOsm38 
Reagents Required 
Method 
Alcoholic Fixatives 
Carnoy’s Fixative 
Reagents Required 
Method 
Methacarn 
Reagents Required 
Method 
Wolman’s Solution 
Reagents Required 
Method 
Acetic Alcohol Formalin 
Reagents Required 
Method 
Picric Acid Fixatives 
Rossman’s Fluid 
Reagents Required 
Method 
Gendre’s Fluid 
Reagents Required 
Method 
Bouin’s Fluid 
Reagents Required 
Method 
Mercuric Fixatives 
Buffered Formaldehyde Sublimate 
Reagents Required 
Method 
Zenker’s Fluid 
Reagents Required 
Method 
Helly’s Fluid 
Reagents Required 
Method 
B5 Fixative 
Reagents Required 
Method 
Susa Fluid 
Reagents Required 
Method