IHC

Question 1

What is the purpose of Epitope/Antigen Retrieval?

Answer 1

To break the cross-linkages that are formed during tissue fixation, thereby exposing the epitope for antibody binding.

Question 2

What are the two main methods of Epitope/Antigen Retrieval?

Answer 2

1. Heat-induced epitope retrieval (HIER)
2. Enzyme-induced epitope retrieval (EIER)

Question 3

What is the key element used in Heat-Induced Epitope Retrieval (HIER)?

Answer 3

High heat.

Question 4

What are two common solutions used in HIER?

Answer 4

Sodium citrate buffer or EDTA.

Question 5

Name three pieces of equipment that can be used for HIER.

Answer 5

Modified pressure cooker, laboratory microwave oven, vegetable steamer, or a circulating water bath. (Any three are correct).

Question 6

What is the key agent used in Enzyme-Induced Epitope Retrieval (EIER)?

Answer 6

Proteolytic enzymes (proteases).

Question 7

Name three examples of enzymes used in EIER.

Answer 7

Pronase, ficin, protease, or pepsin. (Any three are correct).

Question 8

What are the two main purposes of blocking reactions in IHC?

Answer 8

1. To block endogenous peroxidase activity (e.g., in tissues with many red blood cells).
2. To block antibody attachment to highly charged collagen and connective tissues.

Question 9

What is commonly used to block endogenous peroxidase activity, and why is it necessary?

Answer 9

3% hydrogen peroxide (H₂O₂) is used to block the activity of endogenous peroxidase, which is naturally present in tissues like those with many red blood cells, to prevent background staining.

Question 10

In the Direct IHC staining method, what is the antibody directly conjugated to?

Answer 10

An enzyme (e.g., HRP).

Question 11

What is the name of the most common Indirect IHC staining method mentioned?

Answer 11

Avidin-Biotin Complex (ABC) method.

Question 12

In the ABC method, what molecule is the secondary antibody conjugated with?

Answer 12

Biotin (a process called biotinylation).

Question 13

In the ABC method, what is the enzyme (HRP) conjugated to?

Answer 13

Avidin.

Question 14

What is a chromogen?

Answer 14

An electron donor that, when oxidized by an enzyme, provides color to the antigen-antibody reaction site.

Question 15

What is the most commonly used chromogen, and what color does it produce?

Answer 15

3,3’-diaminobenzidine tetrahydrochloride (DAB), which produces a chestnut brown color.

Question 16

What enzyme is DAB a substrate for?

Answer 16

Horseradish Peroxidase (HRP).

Question 17

Besides DAB, name another chromogen used with HRP and the color it produces.

Answer 17

Silver, which produces a black color.

Question 18

What enzyme is used with the chromogens Fast Red and NBT/BCIP?

Answer 18

Alkaline Phosphatase (AP).

Question 19

What color does the chromogen Fast Red produce when used with Alkaline Phosphatase?

Answer 19

Bright fuchsia.

Question 20

What color does the chromogen NBT/BCIP produce when used with Alkaline Phosphatase?

Answer 20

Blue.

Question 21

What are the three main types of IHC staining?

Answer 21

Nuclear, Membranous, and Cytoplasmic.

Question 22

What is the effect of increased cold ischemic time on antigen stability?

Answer 22

It decreases antigen stability.

Question 23

What are the three main factors related to the fixative itself that can affect staining?

Answer 23

The type of fixative used, its temperature, and its pH.

Question 24

Which staining marker is used to check for adequate tissue fixation?

Answer 24

Vimentin staining.

Question 25

What two processing steps are required for optimal IHC staining?

Answer 25

Thorough dehydration and wax infiltration.

Question 26

What is a risk of using high temperatures during tissue processing?

Answer 26

It can damage antigen sites.

Question 27

What type of water should be used in the waterbath during microtomy, and why is this important?

Answer 27

Clean deionized water should be used to prevent contamination or artifacts.

Question 28

Why is it necessary to wear gloves during microtomy?

Answer 28

To avoid squamous cell contamination from the skin.

Question 29

What kind of slides are required for microtomy to ensure tissue sections adhere properly?

Answer 29

Charged (+) slides.

Question 30

What is the recommended section thickness for microtomy?

Answer 30

3-4 microns (3-4µ).

Question 31

How often must a positive control be run for each antibody stain?

Answer 31

Each time the antibody stain is performed.

Question 32

What are two recommended ways to run a positive control?

Answer 32

1. Using a multi-tissue control. 2. Placing it on the same slide as the patient’s specimen.

Question 33

What defines a negative control in IHC staining?

Answer 33

A negative control is run with no primary antibody.