Lars-Inge Larsson

Immunocytochemistry (eBook, PDF)

Theory and Practice

• Format: PDF

Produktbeschreibung

A complete and balanced overview of all aspects of immunocytochemistry is presented providing a clear understanding of their impact on experiment. All available techniques and many diagnostic and research applications are included, as well as practical step-by-step instructions for carrying out recommended methods. Intended for the novice as well as the experienced researchers.

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Produktdetails

• Verlag: Taylor & Francis eBooks
• Seitenzahl: 288
• Erscheinungstermin: 25. November 2020
• Englisch
• ISBN-13: 9781000098693
• Artikelnr.: 60468196

Autorenporträt

Lars-Inge Larsson, D.M.Sc., is Professor of Histochemistry and Head of the Department of Molecular Cell Biology, State Serum Institute, Copenhagen, Denmark. Dr. Larsson received his D.M.Sc. in 1975 at the Department of Histology, University of Lund, Sweden. From 1976 to 1982 he held an appointment as Associate Professor at the Institute of Medical Biochemistry, University of Aarhus, Denmark and was appointed Professor of Histochemistry in 1982. Between 1982 and 1987 he directed the first MRC Unit in Denmark, jointly supported by the Danish MRC and Cancer Society. In 1988 he constructed and was appointed head of the new Department of Molecular Cell Biology at the State Serum Institute in Copenhagen. Dr. Larsson is the author of over 200 scientific articles and books. He received the Boehringer-Ingelheim award in 1982, the Mack-Forster award (European Society for Clinical Investigation) in 1986, and the Anders Jahre and Lundbeck awards in 1987. He is, according to a recent survey made by the Institute of Scientific Information (lSI), among the 250 most cited primary authors in all scientific disciplines. His main area of interest is concerned with studies of neurohormonal peptides and biogenic amines and with the development of histochemical and biochemical methods for the study of these substances. He is the author of a very large number of original articles and reviews on immunocytochemical methodology.

Inhaltsangabe

TABLE OF CONTENTS
Chapter
1 Antibodies and Antisera
I. General Aspects of Antibodies and Antigen
Antibody Interactions
II. Antiserum /Antibody Specificity
A. Methods for Examining the Antiserum /Antibody Specificity
B. Antibodies as Site
or Region
Specific Reagents
C. Methods for Examining the Specificity of Antiserum /Antibody Interaction with Tissue
Bound Antigen: Controls
1. Definitions of Specificity
2. First
Level Controls
a. Causes of Falsely Positive Absorption Controls
i. Presence of Antigen
Binding Complexes
ii. Presence of Antibodies to Contaminating Antigens, Present in the Same Compartments as the Antigen of Interest
iii. Overabsorption of Low
Avidity Antibodies
iv. Destruction of Antigens by Antisera
v. Paradoxical Enhancement of Staining Intensity after Antigen Absorptions
b. Causes of Falsely Negative Absorption Controls
i. Complement
Mediated Binding of Immunoglobulins to Tissue
ii. Basic Peptides and Poly
L
Lysine Bind IgG by a Saturable Mechanism
3. Second
Level or Staining Controls
a. Deletion of all Antibodies
b. Deletion of the Primary Antibody
D. Independent Methods for Establishing Immunocytochemical Specificity
References
Chapter
2 Fixation and Tissue Pretreatment
I. Fixation
A. Introduction
B. Formaldehyde
C. Glutaraldehyde
D. Acrolein
E. Carbodiimide, Imidates, and Benzoquinone (Parabenzoquinone, PBQ)
F. DEPC
G. Osmic Acid or Osmium Tetroxide
H. Precipitating Fixatives
I. Combination Fixatives
J. General Aspects of Fixation
II. Tissue Pretreatment
A. Isolated Cells, Cultured Cells, Imprints, and Tissue Fragments
B. Vibratome® Sectioning
C. Cryostat Sectioning
D. Paraffin and Alternative Light Microscopy Embedding Media
E. Freeze
Drying and Freeze
Substitution
F. Plastic Embedding Techniques
1. Epon®
Araldite® Plastics
2. Polar Resins
a. Lowicryl® K4M
b. LR White
G. Additional Embedding Media
H. Ultracry otomy
I. Comparisons and Recommendations
References
Chapter
3 Immunocytochemical Detection Systems
I. General Aspects
Direct vs. Indirect Techniques
II. Labeled and Unlabeled Antibody Detection Techniques
A. Immunofluorescence
1. Fluorochromes and Labeling Techniques
2. Microscopy and Associated Methods for Fluorescence Detection
3. Fading
4. Counterstains
B. Peroxidase
Based (Labeled and Unlabeled) Methods
1. HRP Cytochemistry
a. Precision
b. Sensitivity and Detection Efficiency: Intensification Methods
c. Alternative Chromogens
d. Specificity: Blocking of Endogenous Peroxidase
Like Activities
2. Immunoperoxidase (Peroxidase
Labeled Antibody) Methods
3. Unlabeled Antibody Methods
a. Immunoglobulin Bridge and PAP Methods
b. Double PAP and Double Bridge Variants
c. The Link Antibody (Link Reagent)
d. PAP
Fab
4. Alternative Immunoenzyme Methods
a. Alkaline Phosphatase
b. Glucose Oxidase
C. Protein A
Based Methods
D. Avidin
Biotin Systems
E. Colloidal Gold
Based Methods
1. General Aspects
2. Preparation of Colloidal Gold Particles
3. Conjugation of Proteins to Colloidal Gold Particles
4. Colloidal Gold Probes in Light Microscopy
a. Light and Fluorescence Microscopic Detection
b. Immunogold
Silver Methods
5. Applications at the Scanning and Transmission Electron Microscopic Level
6. Other Applications of Colloidal Gold
F. Ferritin
Based Methods
G. Other Particulate Markers
H. Radioactive Labeling of Antibodies or Protein A
I. Hybrid Antibodies, Chimeral Antibodies, and Haptenated Antibodies
J. Additional Markers
III. Labeled Antigen Detection Methods
A. Conceptual Origins
B. Radioimmunocytochemistry (RICH)
C. Gold
Labeled Antigen Detection (GLAD)
D. Monoclonal Gold
Labeled Antigen Detection (CLONO
GLAD)
IV. Conclusions and Suggestions
A. Light and Fluorescence Microscopy
B. Postembedding Procedures for TEM
C. Pre
Embedding Procedures for TEM
D. SEM
References
Chapter
4 Section Pretreatment, Epitope Demasking, and Methods for Dealing with Unwanted Staining
I. Section Attachment to Slides and Free
Floating Sections
II. Removal of Embedding Media
III. Pretreatment of Sections to Remove Endogenous Interfering Activities
IV. Demasking of Epitopes
V. Pretreatment, Dilutions, and Application of Primary Antibodies
A. Purification of Antisera and Antibodies
B. Dilutions, Time of Incubation, Sensitivity, and Detection Efficiency
C. Prozone Phenomenon: Implications for Quantitation
D. Temperature
E. Storage of Antisera and Dilutions
F. Application of Antisera to Sections
VI. Buffers and Detergents
A. Buffers
B. Detergents
VII. Special Considerations for Postembedding Immunoelectron Microscopy
VIII. Approaches to Avoid Unspecific Staining
References
Chapter
5 Associated Methods
I. Immunocytochemical Demonstration of Multiple Antigens
A. Adjacent and Mirror Section Methods
B. Elution Double (Multiple) Staining Methods and Sequential Staining Methods
C. Nonelution Double and Multiple Staining Methods for Light Microscopy
1. Direct Immunocytochemical Methods
2. Methods Employing Primary Antisera from Different Species and Species
Specific Second Antibodies
3. Methods Employing Primary Antisera from the Same Species in Indirect Methods
D. Double and Multiple Staining Techniques for Electron Microscopy
1. Direct Immunocytochemical Methods
2. GLAD Method
3. Protein A
Colloidal Gold Method
4. Two
Face Staining Method
5. Primary Antisera from Different Species and Gold
Conjugated Species
Specific Second Antibodies
6. Methods Based on Destruction of Antigen
Combining Sites on Second Antibodies
7. Combinations of Gold
Silver
and Peroxidase
Based Methods
8. Additional Combined Methods and Quantitative Implications
9. Immunogold
Silver Approaches
II. Immunocytochemistry of Living Cells
III. Combinations of Immunocytochemistry with Other Techniques
A. Combinations with Histology
B. Formaldehyde
Induced Fluorescence of Monoamines
C. Autoradiographic Techniques
D. Retrograde Axonal Transport and Microinjection Techniques
IV. Receptor Studies
V. Quantitation
References
Appendix
I. Coupling of Peptide Haptens to Carrier Proteins
A. Carbodiimide Coupling
B. Glutaraldehyde Coupling
II. Purification of Antisera
A. Purification of IgG by Affinity Chromatography and Preparation of F(ab)2 Fragments
B. Coupling of Proteins or Peptides to Cyanogen Bromide
Activated Sepharose® 4B
C. Tissue Powder Absorption
III. Cytochemical Models for Assaying Antibody Specificity and Method Sensitivity and for Screening for Monoclonal Antibodies
A. Peptide Antigens
B. Other Antigens
IV. Fixatives
A. Formaldehyde
Based Fixatives
1. 4% Paraformaldehyde in 0.1 M Sodium Phosphate Buffer, pH 7.3
2. Formaldehyde Fixation at Optimized pH
3. Bouin's Fluid
4. Zamboni's (Stefanini's) Fixative
5. Periodate
Lysine
Paraformaldehyde (PLP)
6. Formol Sublimate
B. Glutaraldehyde
Based Fixatives
1. The Kamovsky
Type Mixtures
2. Acrolein
Glutaraldehyde Combinations
3. Picric Acid
Paraformaldehyde
Glutaraldehyde (PPG)
4. Carbodiimide
Glutaraldehyde
5. Parabenzoquinone
Glutaraldehyde and Parabenzoquinone
Glutaraldehyde
Formaldehyde (PG and PGF)
6. Periodate
Lysine
Paraformaldehyde
Glutaraldehyde (PLPG)
C. Acrolein
D. Diimidoesters
E. Osmic Acid (Osmium Tetroxide)
F. Carbodiimide
G. Parabenzoquinone (Benzoquinone, PBQ)
H. Diethy lpyrocarbonate
I. Camoy's Fixative and Modifications
1. Camoy's Fixative
2. Modified Camoy (MOCA)
3. Methacam and Modified Methacam
J. The Saint
Marie Procedure
V. Tissue Pretreatment and Embedding Procedures
A. Pre
Embedding (Nonembedding) Procedures
1. Isolated Cells, Glands, Tissues, and Monolayer Cultures
a. Dehydration
Rehydration Permeabilization
b. Detergent
Based Permeabilization
c. Controls
2. General Guidelines for Preembedding Staining of Cells Cultured on Plastic Surfaces
3. Pre
Embedding Staining of Cryostat or Vibratome® Sections and Slices
a. Cryostat Sections
b. Vibratome® Sections
4. Pre
Embedding Staining: General Considerations
B. Postembedding Procedures for Electron Microscopy or Light Microscopy
1. Cultured or Isolated Cells
2. Tissue Specimens: Embedding in Nonpolar Resins
3. Tissue Specimens: Embedding in Lowicryl® K4M
Low
Temperature Procedure
4. Tissue Specimens: Embedding in Lowicryl® K4M
Rapid and Intermediate Procedures
5. Tissue Specimens: Ultracryotomy
a. Fixation
b. Washing and Cryoprotection
c. Freezing
d. Sectioning
e. Immunocytochemical Staining
f. Contrasting
C. Postembedding Procedures Primarily for Light Microscopy
1. Standard Paraffin Embedding Methods
2. Cryostat Sectioning
3. Vibratome® Sectioning
4. Embedding in Polyethylene Glycol (PEG)
5. Freeze
Drying and Vapor
Fixation
VI. Labeling of Antibodies
A. Fluorochroming of Proteins
B. Preparation of Colloidal Gold Conjugates
1. Preparation of Colloidal Gold Particles
a. Citrate Method
b. Ascorbate Method
c. White Phosphorus Method
d. Thiocyanate Gold
e. Ultrasonicated Gold
f. The Tannic Acid Method of Slot and Geuze
g. Colloidal Silver
2. Practical Notes on the Preparation of Colloidal Gold and Silver
3. Coupling of Antibodies and Other Reporter Proteins to Colloidal Gold (Silver)
a. Protein Concentration Isotherms
b. Determining Optimal pH for Coupling
c. The Coupling Procedure
d. Testing Gold Conjugate
VII. Postembedding and Nonembedding Staining for Light or Fluorescence Microscopy
A. Pretreatment of Sections and Application of Primary Antiserum
1. Paraffin Sections
2. Epon® or Araldite® Sections
3. Pretreatment of Hydrated Sections
4. Application of Primary Antiserum
5. Rinsing of Sections
B. Indirect Immunofluorescence
C. PAP Procedure and Peroxidase Visualization and Intensification Methods
D. Indirect Immunoperoxidase or Immunoalkaline Phosphatase Staining
E. ABC Method
F. APAAP Method
G. Immunogold
Silver Staining
1. The Silver Nitrate Method
2. The Silver Lactate Method
3. Special Considerations
H. Clono
GLAD Method
I. Radioimmunocytochemistry (RICH)
VIII. Postembedding Staining for Electron Microscopy
A. Pretreatment of Grids and General Considerations
B. Incubation with Primary Antibodies
C. Peroxidase
Antiperoxidase (PAP) Staining
D. Gold
Labeled Antigen Detection (GLAD)
E. Immunogold Staining
F. Protein A
Gold Staining
IX. Methods for Enlarging Gold Particle Size by Silver Enhancement
A. Ultrastructural Autometallography
B. Silver Enhancement with Protecting Colloid
C. Physical Development with L4 Emulsion as Silver Donor
X. Multiple Staining Methods
A. Antibody Elution Technique
B. Formaldehyde Blocking Technique
XI. Abbreviations
References
Index.