Immunohistochemistry

Question 1

What is immunohistochemistry?

What happens to the protein in its natural environment?

Will the staining intensity determine the amount of protein?

Answer 1

◦ A method of detection using antigen-antibody complexes

◦ Immunological localization of a protein in its natural environment.

◦ Staining intensity may NOT determine amount of protein

Question 2

4 methods:

Answer 2

1.) ELISA
2.) Immunoprecipitation
3.) Enzyme assays
4.) Immunohistochemistry

Question 3

ELISA

1.) What type of well-plates is ELISA usually done in?

2.) Describe the direct method of ELISA.

3.) Describe the indirect method of ELISA?

4.) Describe the sandwich method of ELISA?

Answer 3

◦ Usually done in 96-well plates

◦ Direct: labelled primary antibody

◦ Indirect: Labelled secondary antibody

◦ Sandwich: bound between 2 primary antibodies

Question 4

Describe Immunoprecipitation.

Answer 4

◦ Antibodies with substances (usually metals) attached to them bind to antigens and are separated from substance by precipitation (magnets)

Question 5

Describe Enzyme Assays.

Answer 5

◦ Use enzyme and substrates to produce color

Question 6

What is an antigen?

What acts as the best antigen?

What other molecules can also be antigenic?

What are the most common antigens that induce anti-body production by the body?

Answer 6

◦ Antigen – Any substance that can induce a detectable immune response.

• Proteins act as the best antigens.
• Polysaccharides, nucleic acids and other polymers can also be antigenic.
• Most common antigens that induce anti-body production by the body are bacteria and viruses

Question 7

What are antibodies?

What are antibodies used for?

What are the five major classes of antibodies?

Answer 7

General Immunology

◦ Antibody – Also known as immunoglobulins, are proteins that are produced by B lymphocytes in response to antigenic stimulation. Used to identify and neutralize antigens.

◦ Five major classes, each antigenically distinct:
*Immunoglobulin G (IgG)
*Immunoglobulin A (IgA)
*Immunoglobulin M (IgM)
*Immunoglobulin D (IgD)
*Immunoglobulin E (IgE)

Question 8

What shape is the basic Antibody molecule?

What regions does the antibody binds to its specific antigen?

What is the “Y” structure composed of?

Answer 8

• Basic structure is a “Y” shaped molecule.
• Upper arms of the “Y” are regions where the antibody binds to its specific antigen.
• Structure of “Y” is composed of:
• Two identical heavy (long) chains (Gamma, alpha, mu, delta, or epsilon)
  &
• Two identical light (short) chains (Kappa or lambda)

Question 9

Do antibodies bind to long or short regions of their specific antigen?

What is the region on an antigen that antibodies bind to called?

True of false?
Many different antibodies may bind to the same antigen, but each antibody binds to a different epitope.

Answer 9

◦ Antibodies bind to short regions of their specific antigen.

◦ Region on antigen that antibodies bind to is the epitope.

◦ Many different antibodies may bind to the same antigen, but each antibody binds to a different epitope. (True)

Question 10

Polyclonal Antibodies.

What happens when B lymphocyte cells are exposed to antigen?

True or False?
Proliferated B cells clone themselves and each single clone produces an identical antibody.

What do VARIOUS clones produce?

Answer 10

Polyclonal antibodies:

◦ When B lymphocyte cells are exposed to antigen, some of these B Lymphocytes proliferate themselves.

◦ These cells clone themselves and each single clone produces an identical antibody. (TRUE)

◦ But various clones produce antibodies of different class specific to different epitopes on the antigen.

Question 11

Why are polyclonal antibodies difficult to standardize?

Is polyclonal antibody assay usage unlimited or limited?

Answer 11

◦ Because of the variability of the immune response from one animal to another…polyclonal antibodies are difficult to standardize.

◦ Can only be used in a limited number of assays.

Question 12

What event revolutionized the whole science of immunology?

How are monoclonal antibodies prepared? (hint: 3 steps)

Answer 12

◦ Development of monoclonal antibody techniques revolutionized the whole science of immunology.

◦ Prepared by:
1.) Mice are injected with antigen

2.) B lymphocytes are harvested from mouse spleen that is making the desired antibodies

3.) These antibodies are then fused with non-secreting myeloma cells (plasma cell tumor)

Question 13

What does In-vitro fusion of Monoclonal antibodies yield?

Can hybrid cells (hybridoma) be cloned? What does the result in?

Is monoclonal antibody production unlimited or limited?

Answer 13

◦ In vitro fusion yields hybrid cells (hybridoma) that retain the same antibody secreting capability of the B-cell and the immortality of the tumor cells.

◦ These cells can be cloned, which in turn produce antibody identical in molecular structure to the original.

◦ Can be produced in unlimited quantities by

Question 14

What are the advantages of monoclonal antibodies? (hint: there’s 6)

Answer 14

1. High homogenecity
2. Absence of nonspecific antibodies
3. No batch to batch or lot to log variability
4. Purer than polyclonal
5. Display the most desirable attributes
   a) High affinity and selectivity
6. Lack of background staining

Question 15

What is the most important reaction in immunohistochemistry?

Answer 15

The MOST important reaction in immunohistochemistry is the reaction between the target (antigen), and the primary antibody!!!

Question 16

What are the immunohistochemical staining techniques used to detect?

What are the immunohistochemical staining methods? (hint: 5)

Answer 16

Various techniques are used to detect the presence of antigen in the patient’s tissue or the presence of antibody in the patient’s serum.

1.) Direct
2.) Indirect
3.) Unlabeled or Soluble Enzyme Immune Complex
4.) Avidin-Biotin
5.) Polymer

Question 17

Describe the Direct Staining Method

What is the antibody labeled with? (hint: 2 options)

Answer 17

◦ Direct Method: Labeled antibody of known specificity is used to identify antigens in the patient’s tissue.

◦ Antibody is labeled with fluorescent dye (FITC) or labeled with an enzyme for subsequent reaction with a chromogen.

Question 18

Describe the Indirect Staining Method

Is the first antibody labeled or unlabeled?
Is the second antibody labeled or unlabeled?

What is another name for this method?

Answer 18

◦ Uses two antibodies.

First antibody is an unlabeled defined antibody.

The second labeled antibody is used to localize the first.

Also known as “two-step” indirect method.

Question 19

Describe the Indirect Three-Step Staining Method

Answer 19

◦ Uses 3 antibodies. Same as “Two-Step” but with third antibody.

“First antibody is an unlabeled defined antibody.
The second labeled antibody is used to localize the first.”

Question 20

Describe the Unlabeled or Soluble Enzyme Immune Complex Method?

What does it use?

What are these techniques based on?

Answer 20

◦ Unlabeled or Soluble Enzyme Immune Complex: Three step method .

Uses primary antibody, linking or secondary antibody, and soluble enzyme-antienzyme complex.

◦These techniques are based on peroxidase-antiperoxidase technique.

Question 21

Which Avidin-Biotin Methods are currently in use?

What does Avidin have a very high affinity for? Is this binding reversible?

Answer 21

◦ Two Avidin-Biotin techniques in current use:
1.) Avidin-Biotin Complex (ABC)
2.) Labeled Avidin-Biotin (LBC)

• Avidin has a VERY HIGH affinity for Biotin and binding is essentially irreversible.

Question 22

What is followed by the primary antibody in both Avidin-Biotin Methods?

For the ABC method, what comes next after the secondary antibody (linking antibody)?

For the LAB method, what comes next after the secondary antibody?

Answer 22

◦ In both methods the primary antibody is followed by a biotinylated secondary antibody (linking antibody).

◦ For the ABC method, next is the application of a performed avidin-biotin-enzyme complex.

◦ For the LAB method, next is the application of enzyme-labeled avidin.

Question 23

What is Polymer Detection considered?

Polymer enzyme molecules such as HRP or alkaline phosphatase are fused with what?

What are the advantages of Polymer Detection? (hint: theres 2)

Answer 23

◦ Considered the next generation of immunohistochemical staining

◦ Polymer enzyme molecules such as HRP or alkaline phosphatase are fused with the secondary antibody

◦ Advantages:
1.) Monomer technology = greater sensitivity
2.) Shorter run times due to less steps involved

Question 24

What is Immunofluorescence?

What is Fluorochrome?

What can be observed when fluorochrome is attached or conjugated to an antibody?

Answer 24

A Method of Visualization

• Fluorochrome is a dye that absorbs light and then emits its own light at a longer wavelength (called fluorescence).
• When attached or conjugated to an antibody, sites of reaction between antigen and antibody can be observed.

Question 25

What are the most commonly used fluorochromes in Immunofluorescence (IF)?

Answer 25

Most commonly used fluorochromes in IF is FITC and rhodamine (Texas Red).

Question 26

What is Enzyme Immunohistochemistry?

The fundamental principle of enzyme-labeled antibodies is similar to what?

What provides the indicator system to visualize the location of the antibody in enzyme immunohistochemistry?

Which enzymes are used in enzyme histochemistry? Two
most commonly used?

Answer 26

• Method of Visualization

◦ Fundamental principle of enzyme-labeled antibodies is similar to fluorescein-labeled antibodies.

◦ Enzymes (used as markers), in presence of a substrate and a chromogen, provides the indicator system to visualize the location of the antibody.

Variety of enzymes are used, such as:
1.) Alkaline phosphatase (most commonly used)
2.) horseradish peroxidase. (most commonly used)
3.) beta galactosidase.
4.) glucose oxidase.

Question 27

What is Horseradish peroxidase composed of?
What part is the enzyme?
What part is the substrate?

What are some examples of the chromogens that can be used?

What is DAB composed of?

Answer 27

◦ Horseradish peroxidase (enzyme) with Hydrogen peroxide (substrate) with Chromogen.

Chromogens including:
1.) DAB (3,3’-Diaminobenzidine)
- 1% Cobalt Chloride
- Imidazole
- Nickel-sulfate
- Post staining Osmium Tetroxide
OR
2.) AEC (3-amino-9-ethylcarbazole)

Question 28

What is Alkaline phosphatase composed of?
What part is the enzyme?
What part is the substrate?

What are some examples of the chromogens that can be used?

All chromogens noted except WHAT are soluble and require aqueous mounting medium?

What must be used as a counterstain?

Answer 28

◦ Alkaline phosphatase (enzyme) with Naphthol-AS-phosphate (substrate) with Chromogen.

Chromogens including:
- Fast violet red LB
- Fast red TR, or
- Fast blue BBN.

All chromogens noted except DAB are soluble and require aqueous mounting medium.

No alcoholic hematoxylin must be used as counterstain

Question 29

What must be preserved when using the relatively sensitive method called immunofluorescence?

How do most antigens behave in aqueous solutions?

What constitutes the classic preparation of tissue for IF?

Answer 29

◦ Immunofluorescence is a relatively sensitive method, and antigenic reactivity must be preserved.

◦ Most antigens are soluble in aqueous solutions.

◦ Frozen sections of unfixed tissue constitute the classic preparation of tissue for IF.

Question 30

What are the preferred fixatives for Immunostaining? (hint: there’s 6)

What is Zinc formalin noted to do?

Which fixative is advocated for lymph node specimens?

Answer 30

1.) Acetone
2.) B-5 (advocated for lymph node specimens)
3.) Zenker’s
4.) Bouin
5.) Alcohol based (Methacarn)
6.) Zinc formalin

Zinc formalin is noted to preserve immunoreactivity remarkably well and can be used on automatic tissue processors.

Question 31

What does Formalin do in Epitope Enhancement or Retrieval?

What causes excessive cross-linking of proteins?

What does cross-linking result in?

Answer 31

◦ Formalin (aldehyde fixed) tissue compromises immunoreactivity.

◦ Over- fixation causes excessive cross-linking of proteins.

◦ Resulting in antibodies not having access to their respective epitopes.

Question 32

How can detectability antigens in formalin fixed tissue be greatly improved?

Name 2 epitope enhancement (retrieval) methods.

Answer 32

◦ Detectability of many antigens in formalin fixed tissue can be greatly improved with epitope enhancement (retrieval) methods.

◦Two methods used are:
1.) HIER (Heat-induced epitope retrieval)
2.) EIER (Enzyme-induced epitope retrieval)

Question 33

What does HIER stand for?

How is HIER performed?

What negative effects can some HIER methods have?

Answer 33

◦ Heat-induced epitope retrieval

• Heat (microwave) with tissue sections in metallic salt solution, heated to 100℃ (original protocol).
• Some HIER methods can cause morphological damage, loss of tissue, burns and destruction of anti-genicity.

Question 34

What does EIER stand for?

Which epitope retrieval method is the original method?

What are the most common enzymes for digestion? (hint: there’s 5)

The enzyme with what is incubated for a specific time and temperature?

Answer 34

• Enzyme-Induced Epitope Retrieval

◦ EIER is the original epitope retrieval method.

1.) Trypsin
2.) Proteinase K
3.) Chymotrypsin
4.) Pepsin
5.) Pronase

• Enzyme with tissue is incubated at for specific time

Question 35

Proteolytic enzyme digestion usually does what?

If not carefully used, proteolytic enzyme digestion may do what? (hint: 4 things)

Answer 35

Proteolytic enzyme digestion usually reduces nonspecific staining. Only a few antibodies are demonstrated best.

If not carefully used, Proteolytic enzyme digestion
may:
1.) Increase nonspecific staining if not carefully used.
2.) Weaken specific staining
3.) Create false-negative results 4.) Cause fragmentation or loss of tissue sections.

Only a few antibodies are demonstrated best

Question 36

What does Blocking mean?

What is reduced during blocking reactions?

Do immunoperoxidase methods utilize blocking reactions?

Answer 36

◦ Blocking means to “interrupt” any possible reactions with endogenous materials to avoid non-specific staining

• Reduces non-specific background staining *(i.e.: peroxidase, biotin, proteins)

◦ Yes, There can be several types of blocking reactions involved in immunoperoxidase methods.

Question 37

Name 4 Blocking Reactions.

What is the Hydrogen peroxide blocking reaction usually prepared in?
What is the hydrogen peroxide used to block?

What are Low concentrations of Avidin & Biotin solutions used to block?

What are Low concentrations of Casein used to block?

What is Alkaline Phosphatase?

When Endogenous phophatases found in intestines react with IHC subtrates, this causes what?

What is used as the blocking reagent with Alkaline Phosphatase?

Answer 37

1.) Hydrogen peroxide, 2.) Low concentrations of Avidin & Biotin solutions, 3.) Low concentrations of Casein.

1.) Hydrogen peroxide, usually prepared in absolute methanol
* Used to block tissue endogenous peroxidase activity.

2.) Low concentrations of Avidin & Biotin solutions
* Used to block endogenous biotin (vitamin B7).

3.) Low concentrations of Casein
* Used to block nonspecific proteins

4.) Alkaline Phosphatase: Endogenous phophatases found in intestines.

They react with IHC subtrates - causing non specific staining

◦ Levamisole is used as the blocking reagent

Answer 38

Blocking Reactions (example Biotin)
◦Biotin occurs naturally in the body (endogenous).
◦Must be “blocked” so it will not link with Avidin during staining.

Answer 39

Controls

◦Positive Control: Must be run along with each antibody each time immunohistochemical staining is done.

◦Purpose:
•Controls all steps of the analysis
•Trains user for appearance of positive reaction.
•Validates steps of analysis.

Answer 40

Controls

◦Negative Control (Specific & Nonspecific)

◦Purpose:
•Specific: Detects unintended antibody cross-reactivity to cell/cellular components
•Uses same antibody reagent constituted same way
•Nonspecific: Detects unintended background staining
•Uses only diluent prepared with antibody (minus the antibody), known as negative reagent control (confirms patient specimen does not demonstrate staining) endogenous

Answer 41

Quality Control

◦All immuno procedures should be standardized and adhered to as much as possible, following CAP recommendations for laboratory certification.

◦Antibodies must be evaluated and validated with all procedures and results documented accordingly

Answer 42

Quality Control

◦Manufacturer provided information to note:
•Handling instructions
•Expiration date
•Storage requirements
•Suggested working dilution range
•Recommended pretreatment solutions
•Specific antibody information (source, type)

◦Antibodies prediluted or not, must be validated with your laboratory’s procedures.

Answer 43

Antibody Dilution:
◦It is important to dilute the antibodies, as too concentrated of an primary antibody can lead to non-specific overstaining

◦Dilutions are expressed as ratios, for example
•1:100 is the same as “one” to “one-hundred”

◦1:10 dilution means:
•1 part antibody to 9 parts diluent for a total of 10 parts.

Answer 44

Simple Dilutions

◦Prepare a 10ml 1:200 dilution from a stock (undiluted) antibody

•Divide the volume needed by the dilution factor
(10ml / 200 = 0.05ml) to determine the unit volume

•Bring up to volume needed with buffer
(0.05ml stock + 9.95ml buffer)

Answer 45

Antibody Validation

◦Two approaches to validation
•First: using no retrieval, multiple retrieval solutions, and multiple enzyme pretreatments
•Second: Research for information and protocols already tested by the vendor, within journal articles, textbooks, etc.

•Begin with serial dilutions using a standard protocol

•Modify as needed by changing only ONE factor at a

Answer 46

Staining Techniques

◦Basic PAP Immunoperoxidase
◦ABC-Immunoperoxidase
◦Labeled Avidin-Biotin
◦Biotin-Avidin-Horseradish Peroxidase
◦Immunoperoxidase Staining with the three-step indirect method
◦HRP Enzyme-Labeled Polymer Procedure

Answer 47

Staining Techniques

◦Basic PAP Immunoperoxidase Procedure

◦Method uses three reagents:
•Primary antibody
•Secondary antibody
•PAP complex

Answer 48

Staining Techniques

◦ABC-Immunoperoxidase Procedure

◦Method uses three reagents:
•Primary antibody
•Biotinylated secondary antibody
•ABC

Answer 49

Staining Techniques

◦Labeled Avidin-Biotin Technique

◦Method uses three reagents:
•Primary antibody
•Biotinylated secondary antibody
•Avidin conjugated with a marker enzyme

Answer 50

Staining Techniques

◦Biotin-Avidin-Horseradish Peroxidase Procedure

◦This method uses three reagents:
•Primary antibody
•Biotinylated secondary antibody
•Avidin conjugated with horseradish peroxidase

Answer 51

•Staining Techniques

◦Immunoperoxidase Staining with the three-step indirect method

•This is a three stage technique with both the secondary and tertiary antibodies conjugated with peroxidase.

Answer 52

Staining Techniques

◦HRP Enzyme-Labeled Polymer Procedure

•This is a two stage method that uses a primary antibody and a HRP enzyme-labeled polymer secondary.

Answer 53

Key Terms for Neoplasms
◦Neoplasm: cell growth with uncontrolled cell multiplication, can be benign or malignant
◦Anaplasia: lack of differntiation, loss of mature cell morphology. Characteristic of malignancy
◦Metastasis: transfer of disease from primary organ to other parts of body
◦Panels: multiple antibody approach to differentiate one neoplasm from others

Answer 54

Key Terms for Neoplasms
◦Carcinoma: malignancy of epithelial origin
◦Sarcoma: malignancy of connective tissue origin
◦Lymphoma: malignancy of lymphatic system cells
◦Adenocarcinomas: malignancy from glandular epithelial tissues
◦Blastomas: malignancy derived from embryonic type cells

Answer 55

Breast Cancer Markers
◦HER2/neu: Oncogene studied in invasive breast tumors
•Tamoxifen resistant
◦ER/PR: Estrogen receptor and Progesterone receptor antibodies
•Hormone treatment can be helpful with the results of this marker results (Tamoxifen for ER+)

Answer 56

Important antibodies
◦CK7: adenocarcinomas of the breast, lung, ovary, thyroid and pancreas
◦CK20: adenocarcinomas mainly found in the mucous of the ovary and colorectal tumors
◦TTF-1: adenocarcinomas in the thyroid and lung tumors
◦PSA/PSAP: prostate specific markers found in prostate carcinomas

Answer 57

Important antibodies
◦Cytokeratin AE1/AE3: anaplastic tumors in carcinomas and high-grade gliomas
◦HMB-45: melanoma tissue marker
◦GFAP: high grade glioma marker found in astrocytes
◦Vimentin: found in most lymphatic tissue, circulatory tissue, and connective tissues
◦LCA: marker for lymphomas (leukocyte common antigen)

Answer 58

Wilm’s Tumor
◦Small cell neoplastic antibodies used to detect Wilm’s tumor include: Keratin HMW, Vimentin, Desmin, WT-1, CD56.
◦Nephroblastoma, normally found in children

Answer 59

Troubleshooting:

Positive Control AND test specimens are both completely negative.
One or more reagent was omitted, or a non reactive reagent was applied

Answer 60

Troubleshooting:

Test specimens are appropriately stained, but the positive control specimens are either weak or negative

Control simply does not have enough target antigen

Control was treated differently than sample specimen; fixed, processed, or pretreated differently

Answer 61

•Troubleshooting:

Positive control are stained, but your actual sample is weakly stained, or completely negative

Actual sample has a very low amount of target antigen

Answer 62

Troubleshooting:

Negative controls show positive staining (NOT background)

One or more of the reagents are binding to nonspecific antigens.

Avidin enzyme reagent is binding to biotin that is naturally occuring (endogenous)

Answer 63

Troubleshooting:

Some samples within a run are stained well, while others are negative
Sample could be negative, or the run itself was compromised; reagents omitted, programming error, etc.

Answer 64

Troubleshooting:

Excessive nonspecific background staining in test and or samples
Specimen could have dried out, retrieval not done properly, high levels of endogenous biotin