Immunohisto Flashcards
What is histochemistry?
identification and distribution of the chemical constituents in tissues by
means of stains, indicators and microscopy.
• Histochemistry: tissues, cytochemistry: cells
Goals of histochemistry
•Provides color and contrast to microscopic images
• Presentation of normal chemical composition and distribution
• Specificity of the reaction
• Detectability of the reaction product
• Insolubility of the reaction product
Commonly used dyes
•Hematoxylin (basic) – stains nuclei blue
• Eosin (acidic) – stains cytoplasm pink
• Congo red - amyloid
Basic steps of immunohistochem
- fixation and screening
- antigen retrieval
- blocking
- staining with antibodies and detection
- Fixation and sectioning
Tissue embedded in cryoprotective medium and snap-frozen, fixed –
good Ag expression, not so good morphology
or
Formalin-fixed, paraffin-embedded (FFPE) – good morphology
2.Antigen retrieval
if FFPE is used
Improved Ag expression by breaking down formalin-induced Ag cross-
linking, to re-expose Ag epitopes
• Heat-induced (heating slides at pH 6 or 9)
• Enzyme retrieval (proteolytic enzymes i.e. pepsin)
- Blocking
To inactivate endogenous enzymes (e.g. peroxidase in kidney, liver;
alkaline phosphatase in intestine, placenta)
Endogenous antibodies (B cells)
tissue fixation preserves ____ and prevents ______ and ______ of harvested tissues
antigens
autolysis (the destruction of cells or tissues by their own enzymes)
necrosis
why is embedding tissue done
Embedding tissue provides support during sectioning and makes sections
more robust
what is the fixative used for tissue processing, fixation and sectioning
solution 4% formaldehyde (also called paraformaldehyde)
why is it critical to fix or freeze samples quickly and thoroughly after harvesting and ensure that samples are not too large to fix
yes
For non-frozen tissues how is tissue processing done
Fixation- Tissue dehydration in alcohol - paraffin embedding - sectioning -
rehydration - staining with antibodies
where is antigen retrieval performed
on formaldehyde fixed tissue sections to expose antigenic sites and allow antibodies to bind
formaldehyde fixation results in _
Formaldehyde fixation results in protein cross-linking (methylene bridges),
which masks epitopes and can restrict antigen-antibody binding
epitopes
the part of an antigen molecule to which an antibody attaches itself.
antigen retrieval methods break _
Antigen retrieval methods break these methylene bridges and expose
antigenic sites, allowing antibodies to bind
antigen retrieval
heat induced epitope retrieval
Gentle, buffers such as sodium citrate, EDTA, Tris-EDTA, pH 6, 95°C for
10-20 min. Precautions: Heating with microwaves can result in uneven
epitope retrieval due to hot and cold spots. Rigorous boiling can lead to
tissue dissociation from the slide.
antigen retrieval
proteolytic induced epitope retrieval
• Useful for epitopes that are difficult to retrieve.
• pH 7.4, 37°C for 10-15 min, neutral buffer solutions of enzymes such as
pepsin, proteinase K or trypsin.
• Precautions: Enzymatic retrieval can damage morphology of the section –
concentration and timing need to be optimized.
Blocking proteins
• Block with serum or a protein to prevent non-specific binding of antibodies to
tissues or Fc receptors and reduce background and potentially false positive
results.
• Serum matching species of secondary antibody, bovine serum albumin (BSA) are
used
Fc Receptors (FcR) expressed on the cell surface bind to antibodies and facilitate a wide variety of functions related to killing pathogens,
Non-Specific binding (NSB) refers to an occurrence of an antibody binding to unintended proteins, receptors, or transporters.
Binding to the receptor of interest is called specific binding, while binding to the other sites is called nonspecific binding.
Blocking endogenous enzymes
• For enzymatic detection methods, block endogenous enzymes (after incubating
with unconjugated 1° Ab)
Peroxidase blocking: H2O2
(test with 3,3’-diaminobenzidine (DAB) substrate)
Unconjugated antibodies are primary antibodies that are not attached to any substrate
ffpe
Ethanol is used to precipitate proteins during various processes, including purification and crystallization.
Adding alcohol to the solution reduces the hydration of the protein, by removing water and surrounding proteins which leads to aggregation and precipitation.
Alcohol is such a good germ killer because it has the ability to coagulate a germ’s protein.
autolysis (the destruction of cells or tissues by their own enzymes)
In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections
xylenes.” These substances can actually dissolve paraffin,
Clearing is a step that occurs during tissue processing, after water has been removed from a tissue. Xylene is used because wax is not soluble in water.
Paraffin wax is insoluble in water but dissolves in toluene and xylene and melts at 46–68 °C, making it an ideal medium for embedding tissues after they have been dehydrated.
ffpe
In tissue processing, tissue has to be embedded in a medium (such as paraffin) to support it and allow it to be cut without damaging the tissue. Fixed tissue is then dehydrated, removing water from the tissue by escalating grades of alcohol. Alcohol, however, is not miscible with paraffin (something that is miscible can mix well with another substance). As it happens, xylene is highly miscible with paraffin. The reason xylene works so well for tissue processing is that it makes tissues transparent so that paraffin can fully envelop the tissue. And when preparing slides for microscopy, xylene can remove any remaining wax from slides. In this case it is used as clearing agent. This helps with slide staining so that features of the tissue are more easily viewed under a microscope
