Immunohistochemical Staining (IHC)

Question 1

What does immunohistochemistry (IHC) identify?

Answer 1

Biomarkers that guide treatment decisions

Question 2

What are normal results in immunohistochemistry?

Answer 2

Cells appear normal with high levels of maturity and are appropriate for the tissue site

Question 3

What are abnormal results in immunohistochemistry?

Answer 3

Cells appear immature or poorly differentiated, or are found in an inappropriate tissue for their cell type

Question 4

What is a major role of an immunohistochemist?

Answer 4

Troubleshooting for variables introduced by fixation, processing, and tissue types

Question 5

What is an epitope?

Answer 5

The specific site on an antigen where an antibody attaches

Question 6

What is a polyclonal antibody?

Answer 6

A mixture of antibodies from many clones of lymphocytes

Question 7

What is a limitation of polyclonal antibodies?

Answer 7

They are not as selective and can result in non-specific (background) staining

Question 8

What is a monoclonal antibody?

Answer 8

A homogenous population of immunoglobulin directed against a single epitope

Question 9

What are the advantages of monoclonal antibodies?

Answer 9

• High homogeneity
• Absence of nonspecific antibodies
• No batch-to-batch variability
• Unlimited supply

Question 10

How are antibodies used in histology on a tissue section?

Answer 10

Antibodies against characteristic antigens are added to the sample, binding wherever the antigens are present

Question 11

What is the purpose of fixation in immunohistochemistry?

Answer 11

To preserve antigenic reactivity in the tissue

Question 12

What is the recommended method for antigen recovery?

Answer 12

Heat-induced epitope retrieval (HIER)

Question 13

What factors govern the effectiveness of antigen retrieval?

Answer 13

• pH
• Volume of fluid
• Heating time and temperature

Question 14

What are the common retrieval solutions used?

Answer 14

• Sodium citrate buffer (0.01M, pH 6.0)
• EDTA (1mM, pH 8.0)

Question 15

What is the effect of overfixation by formaldehyde?

Answer 15

It can result in the antibody not having access to its epitope, leading to false negative results

Question 16

What is the ideal thickness for tissue sections in IHC?

Answer 16

3-4 microns

Question 17

What is the result of improper positioning of tissue sections on slides?

Answer 17

Areas of incomplete coverage or drying may cause false negative or nonspecific results

Question 18

What is the purpose of automation in immunohistochemistry?

Answer 18

To perform steps of heating, deparaffinization, and antigen retrieval, reducing human error and ensuring consistent temperature and exposure time.

Question 19

What does HIER stand for in immunohistochemistry?

Answer 19

Heat-Induced Epitope Retrieval.

Question 20

What factor is important for nuclear and cell surface antigens during antigen retrieval?

Answer 20

pH.

Question 21

Name three methods of heating used in immunohistochemistry.

Answer 21

• Lab microwave
• Modified pressure cooker
• Circulating water bath.

Question 22

What does EIER stand for in immunohistochemistry?

Answer 22

Enzyme-Induced Epitope Retrieval.

Question 23

What is a proteolytic enzyme used for in EIER?

Answer 23

To expose epitope sites.

Question 24

List three enzymes that can be used in EIER.

Answer 24

• Pronase
• Trypsin
• Pepsin.

Question 25

Fill in the blank: The enzymatic activity is stopped by placing the specimen in cold buffer at _______ prior to processing with antibody.

Answer 25

4°C.

Question 26

What are some disadvantages of the EIER method?

Answer 26

* May increase non-specific staining if not properly used * May weaken specific staining causing false negatives * May cause fragmentation or loss of tissue sections.

Question 27

What is the benefit of combining HIER and EIER?

Answer 27

To enhance the quality of morphology and minimize enzymatic pretreatment.

Question 28

What is a fluorochrome?

Answer 28

A dye that absorbs light and emits its own light at a longer wavelength.

Question 29

What is the principle of immunofluorescence?

Answer 29

Fluorochrome attached to an antibody allows visualization of the reaction sites between antigen and labeled antibody.

Question 30

What are the most common fluorochromes used in immunofluorescence?

Answer 30

* Fluorescein isothiocyanate (FITC) * Rhodamine.

Question 31

True or False: Enzyme IHC is recommended for tumor differentiation.

Answer 31

True.

Question 32

What is the role of chromogen in enzyme IHC?

Answer 32

It provides the indicator system to visualize the location of the antibody.

Question 33

List two commonly used enzymes in enzyme IHC.

Answer 33

* Horseradish peroxidase (HRP) * Alkaline phosphatase (AP).

Question 34

What can happen if hematoxylin containing alcohol is used with AEC or AP chromogens?

Answer 34

The reaction product will be dissolved and removed, leading to false negative results.

Question 35

Fill in the blank: The direct method of immunohistochemistry uses a _______ antibody to identify antigens.

Answer 35

labeled.

Question 36

What is the advantage of the indirect method over the direct method?

Answer 36

More sensitive due to signal amplification and more versatile.

Question 37

What is the purpose of the three-step indirect method?

Answer 37

To further amplify the signal for staining antigens with a limited number of epitopes.

Question 38

What does PAP stand for in immunohistochemistry?

Answer 38

Peroxidase-Anti-Peroxidase.

Question 39

What fixative is used in the basic PAP immunoperoxidase procedure?

Answer 39

10% NBF with epitope enhancement/retrieval.

Question 40

What steps are involved in the basic PAP immunoperoxidase procedure?

Answer 40

* Deparaffinize * Dehydrate * Block endogenous reaction * Apply primary antibody.

Question 41

What is the purpose of the blocking serum in the Basic PAP procedure?

Answer 41

To prevent non-specific binding of antibodies ## Footnote Blocking serum is crucial for reducing background staining.

Question 42

What is the chromogen used in the Basic PAP procedure?

Answer 42

AEC

Question 43

What color indicates a positive reaction in the Basic PAP procedure?

Answer 43

Brick red

Question 44

True or False: If DAB is used as chromogen instead of AEC, a synthetic resin may not be used.

Answer 44

False

Question 45

What are the two techniques used in the Avidin-Biotin method?

Answer 45

* Avidin-biotin complex method (ABC) * Labeled avidin-biotin method (LAB)

Question 46

What is the purpose of pre-treating tissue sections with H2O2 in the Avidin-Biotin method?

Answer 46

To remove any endogenous peroxidase enzyme activity

Question 47

What does DAB reagent produce when it reacts with peroxidase?

Answer 47

An insoluble dark brown precipitate

Question 48

True or False: Positive controls can be commercially prepared slides.

Answer 48

False

Question 49

What should be used to block nonspecific background staining?

Answer 49

Non-immune serum from the same species as the primary antibody

Question 50

What are the two main blocking reactions in most immunoperoxidase methods?

Answer 50

* H2O2 in absolute methanol * Human serum from the same species

Question 51

What is the best practice for daily quality control (QC) in staining?

Answer 51

Review and document results of antibody staining for each day

Question 52

What may cause false positive results due to background staining?

Answer 52

Delayed fixation or inadequate fixation

Question 53

What is the purpose of using a secondary or linking antibody in the Avidin-Biotin method?

Answer 53

To bind to the primary antibody and also to bind ABC

Question 54

What type of antibody has a shorter shelf life and cannot be frozen?

Answer 54

Prediluted antibody

Question 55

What is the ideal storage temperature for most antibodies?

Answer 55

4°C to 8°C

Question 56

What should be done if antibodies are stored at -20°C to -70°C?

Answer 56

Avoid repeat freeze/thaw cycles

Question 57

What is a common cause of overstaining?

Answer 57

Primary and/or secondary antibody concentration too high ## Footnote This can lead to excessive background and false positives.

Question 58

What is the recommended solution for high antibody concentration?

Answer 58

Reduce antibody concentration. Titrate antibodies for optimal dilution ## Footnote Titration helps find the best dilution for accurate results.

Question 59

What should be done if incubation temperature is too high?

Answer 59

Reduce temperature ## Footnote High temperatures can denature antibodies and affect binding.

Question 60

If sections have dried out, what is the solution?

Answer 60

Avoid drying out of tissues ## Footnote Dried sections can lead to compromised staining.

Question 61

What is a potential cause of high background in staining?

Answer 61

Sections inadequately washed at least 3X between steps ## Footnote Insufficient washing can retain excess reagents.

Question 62

What can be done if tissue contains endogenous enzymes like peroxidase?

Answer 62

Block endogenous enzymes prior to incubation of primary antibodies ## Footnote This prevents false positives due to enzyme activity in the tissue.

Question 63

What is the solution if tissue contains endogenous biotin activity?

Answer 63

Block endogenous biotin with avidin/biotin blocking reagent prior to incubating primary antibodies ## Footnote This prevents non-specific binding of antibodies.

Question 64

What technique can reduce non-specific binding of primary antibodies?

Answer 64

Use a higher dilution of primary antibodies ## Footnote Higher dilutions can minimize background staining.

Question 65

What is a recommended action if secondary antibodies are not stored properly?

Answer 65

Store secondary antibodies in the dark ## Footnote Light exposure can degrade antibody effectiveness.

Question 66

What might cause insufficient staining in control and specimen?

Answer 66

Improper epitope retrieval ## Footnote Epitope retrieval is critical for antibody recognition.

Question 67

What is a common issue leading to high background staining?

Answer 67

Absent or insufficient blocking of non-specific binding ## Footnote Blocking agents like non-immune serum are essential.

Question 68

What are some methods used in IHC?

Answer 68

Polyclonal and monoclonal antibodies, immunofluorescence, enzyme IHC ## Footnote Various techniques enhance the visualization of antigens.

Question 69

What does CD 3 identify?

Answer 69

T-Cell lymphoma ## Footnote This antibody is specific for T-cells in lymphomas.

Question 70

What does CD 20 identify?

Answer 70

B-Cell lymphoma ## Footnote CD 20 is a marker for B-cells and is used in lymphoma diagnosis.