Lecture 5: Factors Affecting IHC Staining

Question 1

What is the principle aim of IHC?

Answer 1

Attach the maximum amount of label to the antigen with a minimum amount of nonspecific/background binding

Question 2

Sensitivity

Answer 2

The ability of a detection system to detect the target antigen while using increasingly sparse dilutions of primary antibody

Question 3

Specificity

Answer 3

The ability of an antibody to bind exclusively to its particular antigen (no off-target binding)

Question 4

What is cold ischemic time?

Answer 4

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

Question 5

Why is cold ischemic time critical?

Answer 5

Because autolysis and putrefaction begin as soon as tissue leaves the body, disrupting the tissue that needs to be studied

Question 6

Why is letting the tissue dry out a bad thing?

Answer 6

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

Question 7

Prolonged fixation leads to what?

Answer 7

Excessive protein cross-linking and poor antigen retrieval

This can mask methylene bridges formed during aldehyde fixation

Question 8

Formaldehyde/aldehyde fixative substitutes

Answer 8

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

Question 9

Advantages of Formaldehyde substitutes?

Answer 9

Little to no antigen retrieval needed

Less toxic

Arrive pre-mixed and ready-to-use

Question 10

Disadvantages of Formaldehyde substitutes?

Answer 10

May contain additives that cause false positive or false negative IHC results

Fixation takes longer

Question 11

What is the maximum temperature a sample can experience while maintaining antigenicity?

Answer 11

60C because proteins are altered at high temperatures

This applies to heating in ovens and antigen retrieval

Think of cooking an egg white

Question 12

Why should you avoid additives in your waterbath?

Answer 12

They can cause non-specific staining

Question 13

What else can cause non-specific staining in your water bath?

Answer 13

Bacteria from not using DI water

Floaters from not properly skimming and cleaning your water bath between samples

Question 14

Why should slides be dried upright?

Answer 14

To prevent water or air being trapped under the sections which can cause damage or uneven staining

Question 15

Never go from xylene straight to?

Answer 15

Water because they are immiscible

Use a set of graded alcohols as an intermediary
Xylene to 100%EtOH to 95%EtOH to H2O

Question 16

Why is heat important for antigen retrieval?

Answer 16

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

Question 17

Methods for heat antigen retrieval

Answer 17

microwave oven, water bath, pressure cooker, or autoclave

Question 18

PH and antigen retrieval

Answer 18

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

Question 19

Enzymatic antigen retrieval

Answer 19

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)

Question 20

Heat Retrieval Advantages

Answer 20

better efficiency/quality of protein extraction

Question 21

Enzymatic Retrieval Advantages

Answer 21

generally faster

Question 22

Heat Retrieval Disadvantages

Answer 22

generally takes a longer time compared to enzymatic

Question 23

Enzymatic Retrieval Disadvantages

Answer 23

Can destroy some antigens

Will not work on all antigens

Can over digest/destroy tissue if left on for too long

Question 24

Blocking reagent BSA

Answer 24

Bovine Serum Albumin

Question 25

What is blocking for?

Answer 25

reduce non-specific staining caused by antibodies binding to off target proteins in the tissue

Question 26

How does blocking work?

Answer 26

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.

Question 27

What is blocking serum made of?

Answer 27

Dilute (blood) serum from an animal that is the same species as the secondary antibody

Question 28

When is blocking serum applied?

Answer 28

typically before application of the primary antibody

Question 29

What are enzyme blockers?

Answer 29

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

Question 30

When are enzyme blockers applied?

Answer 30

typically before the antibodies But sometimes after the primary if the block is known to interfere with the IHC reaction by altering sensitive epitopes

Question 31

Blocking endogenous biotin

Answer 31

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

Question 32

Where is endogenous biotin found?

Answer 32

cytoplasm of hepatocytes (liver) proximal tubules in the kidney some tumors

Question 33

Autofluorescence with respect to IF

Answer 33

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

Question 34

Direct IF

Answer 34

Simple, uses antibodies from the same species Less signal, higher cost, less flexibility

Question 35

Indirect IF

Answer 35

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

Question 36

2 most common IHC chromogens

Answer 36

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

Question 37

2 most common IF fluorochromes

Answer 37

FITC fluorescin isothiocyanate, and rhodamine

Question 38

How do fluorochromes work?

Answer 38

the dyes absorb UV light and then emit it at a longer wavelength that is visible to us using a fluorescence microscope

Question 39

Which hematoxylin is preferred for IHC

Answer 39

Mayer's because it does not contain alcohol, esp for AEC detection methods which would be washed out resulting in a false negative

Question 40

Direct IF Advantages

Answer 40

Shorter staining times (good for skin lymphomas)

Question 41

Direct IF Disadvantages

Answer 41

``` Frozen tissue is best Requires dark-field microscopy Poor morphological resolution Autofluorescence Lower signal amplification Higher cost ```

Question 42

Indirect IF Advantages

Answer 42

Greater sensitivity than direct IF More amplified signal than direct IF One secondary antibody can attach to multiple primary antibodies Less expensive

Question 43

Indirect IF Disadvantages

Answer 43

Potential for cross-reactivity | May exhibit high background

Question 44

Direct IHC Advantages

Answer 44

Short and quick | Only uses a primary antibody

Question 45

Direct IHC Disadvantages

Answer 45

Little signal amplification (less sensitive)

Question 46

PAP (peroxidase anti peroxidase, ABC (avidin biotin complex) Indirect Advantages

Answer 46

More sensitive than direct due to signal amplification

Question 47

PAP (peroxidase anti peroxidase, ABC (avidin biotin complex) Indirect Disadvantages

Answer 47

May take longer (more steps)

Question 48

LSAB labeled streptavidin-biotin Indirect Advantages

Answer 48

Same as PAP and ABC But more sensitive No pre-assembly of the ABC complex required

Question 49

LSAB labeled streptavidin-biotin Indirect Disadvantages

Answer 49

Non-specific binding of avidin | Presence of endogenous biotin

Question 50

Polymer IHC Indirect Advantages

Answer 50

Higher specificity and sensitivity than ABC High signal intensity can be done in one step primary antibody/enzyme labeled polymer/chromogen

Question 51

Polymer IHC Indirect Disadvantages

Answer 51

Restricted to a select group of primary antibodies

Question 52

CSA tyramide signal amplification Advantages

Answer 52

Detects small quantities of antigen (high sensitivity) | Enhances the performance of low affinity antibodies

Question 53

CSA tyramide signal amplification Indirect Disadvantages

Answer 53

Time consuming Takes more steps Harder to reproduce Can have high background due to endogenous biotin

Question 54

CSA II Indirect Advantages

Answer 54

Much more sensitive than LSAB, ABC, and CSA tyramide No avidin/biotin reagents, so no reactivity with endogenous biotin

Question 55

CSA II Indirect Disadvantages

Answer 55

Very time consuming