Lecture 5: Factors Affecting IHC Staining Flashcards
What is the principle aim of IHC?
Attach the maximum amount of label to the antigen with a minimum amount of nonspecific/background binding
Sensitivity
The ability of a detection system to detect the target antigen while using increasingly sparse dilutions of primary antibody
Specificity
The ability of an antibody to bind exclusively to its particular antigen (no off-target binding)
What is cold ischemic time?
Time between removing tissue form the body and submerging it in fixative
We want it to be as short as possible-> target is 1 hour in hospital situations
Why is cold ischemic time critical?
Because autolysis and putrefaction begin as soon as tissue leaves the body, disrupting the tissue that needs to be studied
Why is letting the tissue dry out a bad thing?
Negatively impacts cellular morphology, such as nuclear detail/poorly defined chromatin, which is critical for pathologists to read
Increases the risk for non-specific binding or staining artifacts
Prolonged fixation leads to what?
Excessive protein cross-linking and poor antigen retrieval
This can mask methylene bridges formed during aldehyde fixation
Formaldehyde/aldehyde fixative substitutes
fix tissue while avoiding masking epitopes
Can be aqueous or alcohol based
Penetrate slower than formalin, so tissue needs to be trimmed as thin as possible before fixing
Advantages of Formaldehyde substitutes?
Little to no antigen retrieval needed
Less toxic
Arrive pre-mixed and ready-to-use
Disadvantages of Formaldehyde substitutes?
May contain additives that cause false positive or false negative IHC results
Fixation takes longer
What is the maximum temperature a sample can experience while maintaining antigenicity?
60C because proteins are altered at high temperatures
This applies to heating in ovens and antigen retrieval
Think of cooking an egg white
Why should you avoid additives in your waterbath?
They can cause non-specific staining
What else can cause non-specific staining in your water bath?
Bacteria from not using DI water
Floaters from not properly skimming and cleaning your water bath between samples
Why should slides be dried upright?
To prevent water or air being trapped under the sections which can cause damage or uneven staining
Never go from xylene straight to?
Water because they are immiscible
Use a set of graded alcohols as an intermediary
Xylene to 100%EtOH to 95%EtOH to H2O
Why is heat important for antigen retrieval?
Because heating proteins fixed by formaldehyde results in hydrolysis that breaks down protein cross-links, re-exposing previously masked antigens without damaging the fixed tissue
heat “opens up” proteins, thus exposing antigens
Methods for heat antigen retrieval
microwave oven, water bath, pressure cooker, or autoclave
PH and antigen retrieval
pH affects success of retrieval
Optimal pH depends on the solution/kit beings used
Sub-optimal pH can cause tissue to detach from the slide
Enzymatic antigen retrieval
proteolytic (protein cleaving) enzymes break cross-links formed during fixation, “chewing”
Helps improve reagent penetration during subsequent IHC
Effective use is both time (duration) and temperature sensitive (high temp seeds up enzymatic action)
Heat Retrieval Advantages
better efficiency/quality of protein extraction
Enzymatic Retrieval Advantages
generally faster
Heat Retrieval Disadvantages
generally takes a longer time compared to enzymatic
Enzymatic Retrieval Disadvantages
Can destroy some antigens
Will not work on all antigens
Can over digest/destroy tissue if left on for too long
Blocking reagent BSA
Bovine Serum Albumin
What is blocking for?
reduce non-specific staining caused by antibodies binding to off target proteins in the tissue
How does blocking work?
The blocking reagent competes for the non-specific binding sites in the specimen. By binding to those non-specific sites they are no longer available to the antibody for staining.
What is blocking serum made of?
Dilute (blood) serum from an animal that is the same species as the secondary antibody
When is blocking serum applied?
typically before application of the primary antibody
What are enzyme blockers?
They are used to block endogenous activity of HRP horse radish peroxidase and alkaline phosphatase AP, which are the two main detection methods for visualizing antibody binding
we don’t want non-specific pigment developing due to the endogenous presence of these naturally occurring chromogens
When are enzyme blockers applied?
typically before the antibodies
But sometimes after the primary if the block is known to interfere with the IHC reaction by altering sensitive epitopes
Blocking endogenous biotin
Based on high affinity between biotin and streptavidin
Saturate the tissue with free avidin which binds up all the available endogenous biotin, then add free biotin to bind up all the empty receptors on the avidin (4 biotin receptors per avidin).
Nothing reactive is left and non-specific staining is reduced
Where is endogenous biotin found?
cytoplasm of hepatocytes (liver)
proximal tubules in the kidney
some tumors
Autofluorescence with respect to IF
mitochondria and lysosomes naturally fluoresce
Formalin fixation also causes high autofluorescence, which is why using frozen tissue is preferred for IF
This can be reduced by washing paraffin prepped or simply fixed sections in PBS containing sodium borohydride
Direct IF
Simple, uses antibodies from the same species
Less signal, higher cost, less flexibility
Indirect IF
Amplified signal, flexibility through an array of secondary antibody colors, low cost because one secondary can be used with many primaries
More steps so it takes longer, can’t use a primary and secondary from the same species, background may be amplified
2 most common IHC chromogens
DAB (brown) used with horseradish peroxidase system
carcinogenic
AEC(red) also used with horseradish peroxidase, end product is alcohol soluble, so don’t use alcohol based mounting media to coverslip
2 most common IF fluorochromes
FITC fluorescin isothiocyanate, and rhodamine
How do fluorochromes work?
the dyes absorb UV light and then emit it at a longer wavelength that is visible to us using a fluorescence microscope
Which hematoxylin is preferred for IHC
Mayer’s because it does not contain alcohol, esp for AEC detection methods which would be washed out resulting in a false negative
Direct IF Advantages
Shorter staining times (good for skin lymphomas)
Direct IF Disadvantages
Frozen tissue is best Requires dark-field microscopy Poor morphological resolution Autofluorescence Lower signal amplification Higher cost
Indirect IF Advantages
Greater sensitivity than direct IF
More amplified signal than direct IF
One secondary antibody can attach to multiple primary antibodies
Less expensive
Indirect IF Disadvantages
Potential for cross-reactivity
May exhibit high background
Direct IHC Advantages
Short and quick
Only uses a primary antibody
Direct IHC Disadvantages
Little signal amplification (less sensitive)
PAP (peroxidase anti peroxidase, ABC (avidin biotin complex) Indirect Advantages
More sensitive than direct due to signal amplification
PAP (peroxidase anti peroxidase, ABC (avidin biotin complex) Indirect Disadvantages
May take longer (more steps)
LSAB labeled streptavidin-biotin Indirect Advantages
Same as PAP and ABC
But more sensitive
No pre-assembly of the ABC complex required
LSAB labeled streptavidin-biotin Indirect Disadvantages
Non-specific binding of avidin
Presence of endogenous biotin
Polymer IHC Indirect Advantages
Higher specificity and sensitivity than ABC
High signal intensity can be done in one step
primary antibody/enzyme labeled polymer/chromogen
Polymer IHC Indirect Disadvantages
Restricted to a select group of primary antibodies
CSA tyramide signal amplification Advantages
Detects small quantities of antigen (high sensitivity)
Enhances the performance of low affinity antibodies
CSA tyramide signal amplification Indirect Disadvantages
Time consuming
Takes more steps
Harder to reproduce
Can have high background due to endogenous biotin
CSA II Indirect Advantages
Much more sensitive than LSAB, ABC, and CSA tyramide
No avidin/biotin reagents, so no reactivity with endogenous biotin
CSA II Indirect Disadvantages
Very time consuming
