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Immunohistochemistry Fall 2020 > Lecture 5: Factors Affecting IHC Staining > Flashcards

Lecture 5: Factors Affecting IHC Staining Flashcards

1
Q

What is the principle aim of IHC?

A

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

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2
Q

Sensitivity

A

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

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3
Q

Specificity

A

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

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4
Q

What is cold ischemic time?

A

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

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5
Q

Why is cold ischemic time critical?

A

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

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6
Q

Why is letting the tissue dry out a bad thing?

A

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

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7
Q

Prolonged fixation leads to what?

A

Excessive protein cross-linking and poor antigen retrieval

This can mask methylene bridges formed during aldehyde fixation

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8
Q

Formaldehyde/aldehyde fixative substitutes

A

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

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9
Q

Advantages of Formaldehyde substitutes?

A

Little to no antigen retrieval needed

Less toxic

Arrive pre-mixed and ready-to-use

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10
Q

Disadvantages of Formaldehyde substitutes?

A

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

Fixation takes longer

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11
Q

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

A

60C because proteins are altered at high temperatures

This applies to heating in ovens and antigen retrieval

Think of cooking an egg white

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12
Q

Why should you avoid additives in your waterbath?

A

They can cause non-specific staining

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13
Q

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

A

Bacteria from not using DI water

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

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14
Q

Why should slides be dried upright?

A

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

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15
Q

Never go from xylene straight to?

A

Water because they are immiscible

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

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16
Q

Why is heat important for antigen retrieval?

A

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

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17
Q

Methods for heat antigen retrieval

A

microwave oven, water bath, pressure cooker, or autoclave

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18
Q

PH and antigen retrieval

A

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

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19
Q

Enzymatic antigen retrieval

A

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)

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20
Q

Heat Retrieval Advantages

A

better efficiency/quality of protein extraction

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21
Q

Enzymatic Retrieval Advantages

A

generally faster

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22
Q

Heat Retrieval Disadvantages

A

generally takes a longer time compared to enzymatic

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23
Q

Enzymatic Retrieval Disadvantages

A

Can destroy some antigens

Will not work on all antigens

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

24
Q

Blocking reagent BSA

A

Bovine Serum Albumin

25
Q

What is blocking for?

A

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

26
Q

How does blocking work?

A

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.

27
Q

What is blocking serum made of?

A

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

28
Q

When is blocking serum applied?

A

typically before application of the primary antibody

29
Q

What are enzyme blockers?

A

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

30
Q

When are enzyme blockers applied?

A

typically before the antibodies

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

31
Q

Blocking endogenous biotin

A

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

32
Q

Where is endogenous biotin found?

A

cytoplasm of hepatocytes (liver)

proximal tubules in the kidney

some tumors

33
Q

Autofluorescence with respect to IF

A

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

34
Q

Direct IF

A

Simple, uses antibodies from the same species

Less signal, higher cost, less flexibility

35
Q

Indirect IF

A

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

36
Q

2 most common IHC chromogens

A

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

37
Q

2 most common IF fluorochromes

A

FITC fluorescin isothiocyanate, and rhodamine

38
Q

How do fluorochromes work?

A

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

39
Q

Which hematoxylin is preferred for IHC

A

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

40
Q

Direct IF Advantages

A

Shorter staining times (good for skin lymphomas)

41
Q

Direct IF Disadvantages

A 
Frozen tissue is best
Requires dark-field microscopy
Poor morphological resolution
Autofluorescence
Lower signal amplification
Higher cost
42
Q

Indirect IF Advantages

A

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

43
Q

Indirect IF Disadvantages

A

Potential for cross-reactivity

May exhibit high background

44
Q

Direct IHC Advantages

A

Short and quick

Only uses a primary antibody

45
Q

Direct IHC Disadvantages

A

Little signal amplification (less sensitive)

46
Q

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

A

More sensitive than direct due to signal amplification

47
Q

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

A

May take longer (more steps)

48
Q

LSAB labeled streptavidin-biotin Indirect Advantages

A

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

49
Q

LSAB labeled streptavidin-biotin Indirect Disadvantages

A

Non-specific binding of avidin

Presence of endogenous biotin

50
Q

Polymer IHC Indirect Advantages

A

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

51
Q

Polymer IHC Indirect Disadvantages

A

Restricted to a select group of primary antibodies

52
Q

CSA tyramide signal amplification Advantages

A

Detects small quantities of antigen (high sensitivity)

Enhances the performance of low affinity antibodies

53
Q

CSA tyramide signal amplification Indirect Disadvantages

A

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

54
Q

CSA II Indirect Advantages

A

Much more sensitive than LSAB, ABC, and CSA tyramide

No avidin/biotin reagents, so no reactivity with endogenous biotin

55
Q

CSA II Indirect Disadvantages

A

Very time consuming

Immunohistochemistry Fall 2020 (9 decks)

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