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Manual of Histological Techniques Santosh Kumar Mondal

CHAPTER 1:Tissue Fixation and Fixatives
- INTRODUCTION
- CLASSIFICATION OF FIXATIVES
- REACTIONS OF FIXATIVES WITH PROTEINS
- GENERAL PRINCIPLES OF FIXATION
  - Aldehyde (Formaldehyde/10% Formalin/Glutaraldehyde)
- ROUTINE FORMALIN FIXATIVES
  - Chemical Composition of Different Formaldehyde Containing Fixatives
- REMOVAL OF FORMALIN PIGMENT
- PROTEIN DENATURING AGENTS AS FIXATIVE
- MERCURY FIXATIVES
  - Zenker's Fluid
  - Helly's Fluid
- PICRIC ACID FIXATIVES
  - Bouin's Fluid
  - Brasil's Alcoholic Picro-formol Fixative
- FIXATION OF TISSUES FOR ELECTRON MICROSCOPY
- FACTORS INVOLVED IN TISSUE FIXATION
- SECONDARY FIXATION (POST-FIXATION/POST-CHROMATIN)
CHAPTER 2:Tissue Processing, Microtomy and Paraffin Sections
- TISSUE PROCESSING, MICROTOMY AND PARAFFIN SECTIONS
- DEHYDRATION
- CLEARING
- IMPREGNATION
  - Vacuum Impregnation
    - Apparatus
    - Procedure
- EMBEDDING
- AUTOMATED TISSUE PROCESSING
  - Overnight Schedule (Automation Technique)
- ROTARY MICROTOME
- MICROTOME KNIVES
- KNIFE MATERIALS
- KNIFE SHARPENING
  - Hand Sharpening
  - Stropping
- PARAFFIN SECTION CUTTING
- SECTION ADHESIVES
  - Mayer's Egg Albumen-Glycerol
  - Gelatin in Floating Out Bath
- CUTTING HARD TISSUES
- BLOCKING OUT MOLDS (SHAPES, CUPS)
- MOUNTING OF SECTIONS
  - Functions of Mounting Media
CHAPTER 3:Cryostat and Frozen Section
- ADVANTAGE OF FROZEN AND CRYOSTAT SECTIONS
- THE CRYOSTAT (MACHINE)
- FREEZE DRYING
CHAPTER 4:Staining of Tissues—Basic Concept
- FACTORS CONTRIBUTING TO AFFINITIES BETWEEN DYE AND TISSUES
- POLYCHROMASIA
- THEORY OF STAINING
  - Physical Theories
  - Chemical Theories
- STAINS AND DYES
CHAPTER 5:Routine Hematoxylin and Eosin Stain
- PRINCIPLE OF ALUM HEMATOXYLIN STAINING
CHAPTER 6:Carbohydrates
- INTRODUCTION
  - Acidic Mucin
  - Neutral Mucin
- FIXATIVE FOR MUCINS
- CHOICE OF STAINING METHOD
- METACHROMASIA AND MUCIN
- GLYCOGEN
  - Choice of Fixative
  - Embedding Medium
- USE OF ENZYMES
- PERIODIC ACID-SCHIFF REACTION AND STAIN
- SOUTHGATE'S MUCICARMINE METHOD (MAYER, 1896; MODIFIED BY SOUTHGATE, 1927)
  - Composition of Staining Solution
  - Best Carmine Method (Best 1906)
CHAPTER 7:Lipids
- INTRODUCTION
- CLASSIFICATION OF LIPIDS
- CLASSIFICATION OF LIPIDS BASED ON POLAR AND NONPOLAR GROUPS
- FIXATION OF LIPIDS
- IDENTIFICATION OF LIPIDS
- CONTROL SECTIONS
CHAPTER 8:Proteins and Nucleic Acid Staining
- INTRODUCTION
- HISTOPHYSICAL METHODS
- AMINO ACID HISTOCHEMICAL METHODS
- ENZYME HISTOCHEMICAL METHODS
- IMMUNOHISTOCHEMICAL METHODS
- FLUORESCENT METHODS
- NUCLEIC ACIDS
- DEOXYRIBONUCLEIC ACID
- RIBONUCLEIC ACID
- RNA STAINING
CHAPTER 9:Connective Tissue Staining
- INTRODUCTION
- CELLULAR COMPONENT
- FIBROBLASTS
  - Functions
- MACROPHAGES OR HISTIOCYTES
  - Functions
  - Distribution in the Body
- PLASMA CELLS
  - Functions
- MAST CELL
  - Functions
- PIGMENT CELLS
- RETICULAR CELLS
  - Functions
- FAT CELLS
- INTERCELLULAR SUBSTANCE
- AMORPHOUS ELEMENT
- FIBROUS ELEMENT
- FIBRIN AND FIBRINOID MATERIALS
  - Fibrin
  - Fibrinoid Materials
- MASSON TRICHROME STAIN
- TAKE HOME MESSAGE
CHAPTER 10:Pigments and Minerals
- INTRODUCTION
- HEMOGLOBIN
  - Stains
- HEMOSIDERIN
  - Stains
- BILE PIGMENTS
  - Stains
- PORPHYRIN PIGMENTS
- MELANIN PIGMENTS
  - Stains
- LIPOFUSCINS
  - Stains
- DUBIN-JOHNSON PIGMENTS
- PSEUDOMELANOSIS (MELANOSIS COLI)
  - Stain
- HAMAZAKI-WESENBERG BODIES
- IRON
  - Stains
- CALCIUM
  - Stains
- COPPER
  - Stains
- LEAD
  - Stains
- URIC ACID AND URATES
  - Stains
- CARBON
- SILICA
- ASBESTOS
- SILVER
  - Stains
- TATTOO PIGMENT
- FORMALIN PIGMENTS
  - Removal of Formalin Pigment
- MERCURY PIGMENTS
  - Removal of Mercury Pigment
- CHROMIC OXIDE
  - Removal of Chromic Oxide
- MALARIAL PIGMENT (HEMOZOIN PIGMENT)
  - Removal of Malarial Pigment
- SCHISTOSOME PIGMENT
- PERL'S PRUSSIAN BLUE REACTION (FOR FERRIC IRON)
  - Fixative
  - Sections
  - Principle of the Technique
  - Composition of Staining Solution
  - Positive Control Section
  - Staining Method
  - Results
- TIRMANN-SCHMELZER'S TURNBULL BLUE TECHNIQUE (FOR FERROUS AND FERRIC IRON)
  - Staining Method
  - Results
- HUKILL AND PUTT'S METHOD (FOR FERROUS AND FERRIC IRON)
  - Composition of Staining Solution
  - Staining Method
  - Results
- MODIFIED FOUCHET TECHNIQUE FOR BILE PIGMENTS
  - Composition of Staining Solution
  - Staining Method
  - Result
- GEMELIN TECHNIQUE FOR BILE PIGMENTS
  - Staining Method
  - Results
- MASSON-FONTANA METHOD FOR MELANIN
  - Fixatives
  - Sections
  - Composition of Staining Solution
  - Staining Method
  - Results
- SCHMORL'S REACTION FOR MELANIN
  - Composition of Staining Solution
  - Staining Method
  - Results
- LONG ZIEHL-NEELSEN METHOD FOR LIPOFUSCIN
  - Staining Method
  - Results
- VON KOSSA'S TECHNIQUE FOR CALCIUM
  - Staining Method
  - Result
- ALIZARIN RED S METHOD FOR CALCIUM
  - Staining Solution
  - Staining Method
  - Result
- MODIFIED RHODAMINE TECHNIQUE FOR COPPER
  - Composition of Staining Solution
  - Staining Method
  - Results
- MALLORY AND PARKER'S METHOD FOR LEAD AND COPPER
  - Composition of Staining Solution
  - Staining Method
  - Results
CHAPTER 11:Bone and Decalcification
- INTRODUCTION
- BONE TECHNIQUES
- SPECIMENS AND BIOPIES
- FIXATIVES FOR BONE SPECIMENS
- SAWS IN BONE CUTTING
- DECALCIFICATION
- WEAK ACIDS (FORMIC, PICRIC, ACETIC ACID)
- CHELATING AGENTS (EDTA)
- ION EXCHANGE RESINS
- ELECTROPHORETIC METHOD OF DECALCIFICATION
- SURFACE DECALCIFICATION
- FACTORS OF DECALCIFICATION PROCESS
- PLASTIC OR SYNTHETIC RESIN EMBEDDING
- METHYL METHACRYLATE EMBEDDING PROCEDURE
CHAPTER 12:Nervous System
- INTRODUCTION
- NEURONS
- GLIAL CELL
- MENINGES
- MYELIN
- NISSL SUBSTANCE (TIGROID OR CHROMIDIAL SUBSTANCES)
- CRESYL FAST VIOLET STAIN (NISSL SUBSTANCE)
- LUXOL FAST BLUE – CRESYL VIOLET FOR MYELIN
- PAGE'S SOLOCHROME CYANINE TECHNIQUE (FOR MYELIN)
- GLEES AND MARSLAND'S MODIFICATION OF BIELSCHOWSKY'S METHOD
- EAGER'S METHOD FOR DEGENERATING AXONS
- MODIFIED BIELSCHOWSKY METHOD (FOR NEUROFIBRILLARY TANGLES AND PLAQUES)
CHAPTER 13:Some Special Stains
- AMYLOID
- STAINING FOR AMYLOID
  - Diagnosis
  - Choice of Procedure and Fixative
- WHAT IS BIREFRINGENCE?
  - Why does Amyloid Show Birefringence with Certain Dyes like Congo Red?
  - Why does Amyloid Show Metachromasia with Certain Dyes like Methyl Violet?
- ALKALINE CONGO RED TECHNIQUE
- THIOFLAVIN T METHOD
- LENDRUM'S TECHNIQUE (METACHROMATIC STAIN)
  - Results
  - Points to Remember
- GRAM-TWORT STAIN
- ZIEHL-NEELSEN METHOD
- MODIFIED WADE-FITE METHOD FOR M. LEPRAE AND NOCARDIA
- GIMENEZ METHOD FOR HELICOBACTER PYLORI
- GIEMSA STAIN FOR PARASITES
- PHLOXINE-TARTRAZINE TECHNIQUE
CHAPTER 14:Enzyme Histochemistry and Histochemical Stains
- ENZYMES
- HISTOCHEMISTRY
- ENZYME HISTOCHEMISTRY
  - Fixatives
- PRINCIPLES OF ENZYME HISTOCHEMISTRY
- CLASSICAL HISTOCHEMICAL REACTIONS
- ENZYME TYPES
- TYPES OF HISTOCHEMICAL REACTION
- SKELETAL MUSCLE BIOPSY
- ADENOSINE TRIPHOSPHATASE (ATPASE)
- CYTOCHROME OXIDASE
- PHOSPHORYLASE
- DETECTION OF NERVES AND GANGLIA IN SUSPECTED HIRSCHSPRUNG'S DISEASE
  - Acetylcholinesterase
- CONCLUSION
- AGYROPHIL STAINING
- ARGENTAFFIN STAINING (MASSON- FONTANA METHOD)
CHAPTER 15:Immunohistochemistry
- DEFINITION
- ANTIGEN
- EPITOPES
- ANTIBODY
- SHORT HISTORY
- DIFFERENT TECHNIQUES
- LABELED DIRECT METHOD (TRADITIONAL METHOD)
- UNLABELED ENZYME-ANTIENZYME COMPLEX METHOD (PEROXIDASE- ANTI-PEROXIDASE COMPLEX METHOD)
  - Disadvantages
  - (Strept) Avidin-Biotin Method
- TECHNICAL ASPECTS OF IMMUNOHISTOCHEMISTRY
- CYTOLOGICAL PREPARATIONS
- ADHESIVE FOR HISTOLOGIC SECTIONS
- ANTIGEN RETRIEVAL TECHNIQUES
- HEAT MEDIATED ANTIGEN RETRIEVAL
- PREPARATION OF PARAFFIN WAX SECTIONS FOR IHC
  - Points to Remember
- IHC STAINING PROTOCOL
  - (Strept) Avidin-Biotin Technique
  - Polymer Technique (Dako EnVision)
- MULTIPLE STAINING
CHAPTER 16:Cell Block and Diagnostic Cytopathology
- INTRODUCTION
- PREPARATION OF CELL BLOCK USING AGAR
- PREPARATION OF CELL BLOCK USING THROMBIN AND PLASMA
- POINTS TO REMEMBER
- USE OF CELL BLOCK
- NORMAL SALINE RINSE METHOD
- FIFTY PERCENT ETHANOL RINSE METHOD
- TISSUE COAGULUM CLOT METHOD
- CELLIENTTM AUTOMATED CELL BLOCK SYSTEM
- SHANDON CYTOBLOCK METHOD
- FIXATIVES USED IN CBS
- EMBEDDING AND PARAFFIN SECTIONS
- SHORT SCHEDULE FOR AUTOMATIC TISSUE PROCESSING FOR CELL BLOCKS (13 HOURS)
CHAPTER 17:Plastic/Resin Embedding Media
- INTRODUCTION
- CLASSIFICATION OF PLASTIC MEDIA
  - Composition
- EPOXY RESINS/PLASTICS
- ACRYLIC RESINS/PLASTICS
- APPLICATION OF ACRYLIC PLASTICS
- POLYESTER PLASTICS
- PROCESSING AND EMBEDDING FOR METHYL METHACRYLATE
  - Reagents (Solution)
  - Processing and Embedding Process
- PROCESSING AND EMBEDDING FOR LR WHITE
  - Fixative
  - Processing and Embedding
- PROCESSING AND EMBEDDING FOR GLYCOL METHACRYLATE
- SECTIONING CUTTING OF ACRYLIC RESIN/PLASTIC EMBEDDED SPECIMENS
- STAINING OF ACRYLIC RESIN/PLASTIC SECTIONS
  - H and E Staining Protocols
- BASIC TECHNIQUES FOR EMBEDDING AND SECTIONING PLASTICS/RESINS
CHAPTER 18:Electron Microscopy
- INTRODUCTION
- TYPES OF ELECTRON MICROSCOPES
- RENAL DISEASES
- NEOPLASMS
- METABOLIC DISEASES
- INFECTIOUS DISEASE
- GENETIC DISEASES
- SKELETAL MUSCLE BIOPSY
- PERIPHERAL NERVE BIOPSY
- DISEASES OF OBSCURE NATURES
- LIMITATIONS OF ELECTRON MICROSCOPY
CHAPTER 19:Immunofluorescence Techniques
- DEFINITION
- FLUORESCENCE
- BRIEF HISTORY
- TECHNICAL ASPECTS
- PROCEDURE FOR FROZEN TISSUE SECTIONS
  - Points to Remember
- DIRECT IMMUNOFLUORESCENCE STAINING METHOD (FIG. 1A)
  - Points to Remember
- INDIRECT IMMUNOFLUORESCENCE STAINING METHOD (FIG. 1B)
- FLUORESCENT MICROSCOPE
- DIAGNOSTIC USE OF IMMUNOFLUORESCENCE
CHAPTER 20:Museum Technique
- INTRODUCTION
- RECEPTION OF THE SPECIMEN
- PREPARATION OF THE SPECIMEN
- FIXATION IN FIXATIVE
  - Kaiserling I Solution
- RESTORATION OF COLOR
  - Kaiserling II Solution
- PRESERVATION IN MOUNTING FLUID
- PRESENTATION OF THE SPECIMEN
- PREPARATION OF MUSEUM JARS OR BOXES (PERSPEX)
- MOUNTING IN PERSPEX JARS OR ROUTINE MOUNTING
- MOUNTING IN GLASS JARS
- GELATIN EMBEDDING
CHAPTER 21:Light Microscopy
- INTRODUCTION
- THE MICROSCOPE
- COMPONENT OF A LIGHT MICROSCOPE
- LIGHT SOURCE
- ROUTINE CARE AND MAINTENANCE
  - Routine Care of Microscope
  - Cleaning of Lenses
CHAPTER 22:Molecular Diagnostic Techniques and its Applications
- INTRODUCTION
- SPECIMEN REQUIREMENTS
- DNA ANALYSIS
- RNA ANALYSIS
- PROTEIN ANALYSIS
- INTERPHASE CYTOGENETICS
- MICRODISSECTION
- POLYMERASE CHAIN REACTION (PCR)
  - Procedure
  - Variants of PCR
- APPLICATIONS OF PCR
- DNA MICROARRAYS
CHAPTER 23:In Situ Hybridization and Fluorescent In Situ Hybridization
- INTRODUCTION
- METHODS OF IN SITU HYBRIDIZATION
- IN SITU HYBRIDIZATION (ISH) PROTOCOL
- RESULT
- APPLICATIONS OF ISH
- INTRODUCTION
- BASIC STEPS OF FISH
- PROBES
- LABELING IN FISH
- TISSUE TYPES
- FISH PROCEDURES (GENERAL/COMMON THINGS)
- REAGENTS
- METAPHASE SCORING CRITERIA
  - Reasons for False-negative (Wrong) Results
- SINGLE DAY FISH PROTOCOL (SIGMA– ALDRICH)
APPENDIX:Appendix
- LENGTH
- MASS
- PREPARATION OF SOLUTION
- MOLAR SOLUTION (M)
- PREPARATION OF SOME USEFUL SOLUTIONS
- PREPARATION OF SOME USEFUL BUFFER SOLUTIONS
  - Buffers
Glossary

INDEX

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Chapter Notes