Immunihistochem

Question 1

1. combination of biochem and histology
2. routinely used for the identification of specific or highly selective cellular
   epitopes or antigens in frozen or paraffin-embedded tissues
3. detect organism in cytologic preparations
   ***
4. principle of IH

Answer 1

1. Histochemistry
2. Immunohistochemistry
3. Immunocytochemistry
4. antigen-antibody interactions

Question 2

General Principles:
1. Antibodies belong to the class of serum proteins known
2. most commonly used antibody for immunocytochemistry
3. the structural part of the antigen that reacts with an antibody

Answer 2

1. immunoglobulins
2. IgG
3. epitope

Question 3

1. produced by immunizing an animal with a purified specific molecule that contains the antigen of interest, and collecting immunoglobulin-rich serum
   - purified specific molecule
   - most frequently used animal for the production of polyclonal antibodies
   - when human antigen is given to rabbit, they will produce ____
   - ____ is collected from the lab animal
2. will produce numerous clones of plasma cells (polyclonal) that in turn will produce the antibody; products of an individual clone of plasma cells
   - clone of plasma cells that produce the same type of antibody
   - currently used almost exclusively for the production of monoclonal antibodies
   - can be carried out in culture medium or by transplantation of the hybridoma into the peritoneal cavity of syngeneic mice from where the antibodies are harvested.

Answer 3

1. Polyclonal Antibodies
   - immunogen
   - rabbit
   - anti human antibody
   - Immunoglobulin-rich serum
2. Monoclonal Antibodies
   - Hybridoma
   - mice
   - Propagation

Question 4

Preparing for IHC
1. fixed for a few seconds in ____ or _____
2. adhesive used
3. may done on _____ & ____
4. breakdown of formalin cross linking

Answer 4

1. absolute methanol or acetone
2. Poly-L-lysine
3. Antigen Retrieval

Question 5

ANTIGEN RETRIEVAL:

_______: pre-treated with enzymes to break down formalin cross-linking, unmask and allow certain antigenic sites to be exposed

most common enzymes used
1. _____
- uses
2. _____
- uses
***
1. adjusted to pH ____with ______
2. preheated at ___ in distilled water
3. especially useful for demonstrating (3)

Answer 5

Proteolytic Enzyme Digestion

most common enzymes used
1. TRYPSIN
- 0.1% trypsin in 0.1 % calcium chloride
2. PROTEASE
- 0.05 to 0.1% protease
***
1. pH 7 .8 with sodium hydroxide
2. 37C
3.
- heavy chain immunoglobulins
- complement
- specific antigens

Question 6

ANTIGEN RETRIEVAL:

_______: pretreatment method used to improve staining results

1. ____ breaks the crosslinks that bind surrounding proteins or peptides to the antigen
2. the higher the temp = more effective the recovery of _____
3. uneven or inconsistent which results in a lack of reproducibility
4. uniform and consistent heat distribution.

Answer 6

Heat-induced epitope retrieval

1. thermal energy
2. epitope
3. microwave
4. pressure cooker

Question 7

ANTIGEN RETRIEVAL:

_______: relatively new technique that involves the boiling of formalin-fixed deparaffinized sections in certain solutions

1. certain solutions are (3)
   - ____ (pH ____)
   - ____ (pH ____)
   - ____ (pH ____)
2. optimal length of exposure to heat may vary from
3. most satisfactory time period appears to be
4. Fibrous and fatty tissues tend to detach from the slide which can be prevented by mounting the sections on slides with a strong adhesive like _____

Answer 7

Microwave Antigen Retrieval

1. certain solutions are (3)
   - 0.01 M-citrate buffer (pH 6.0)
   - EDTA at pH 8.0
   - Tris EDTA (pH or 10.0)
2. 10-60mins
3. 20mins
4. Vectabond

Question 8

ANTIGEN RETRIEVAL:

_______: another alternative that appears to be less time consuming and allows for more consistent recovery of many antigens

1. In the large batch microwave oven technique, heating temperature is not uniformly distributed and slides are subjected to “____” and “_____” resulting in inconsistent antigen recovery.

Answer 8

Pressure Cooker Antigen Retrieval

1. “hot spots” & “cold spots”

Question 9

CONTROLS

1. It is always advisable to use, a section that is known and proven to contain the antigen in question because absence of staining in a test section does not necessarily mean that the antigen is absent in the tissue being studied
2. can be done using a parallel section from can be done using a parallel section from
3. eliminates the variable of tissue fixation between specimens and controls but it contains the target antigen, not only in the tissue elements under investigation, e.g. tumors, but also in adjacent normal tissue element
   - aka
   - one example which is in both melanoma and normal tissue

Answer 9

1. Positive Control
2. Negative Control
3. Internal Tissue Control
   - “built in” control
   - S-100 protein

Question 10

LABELLING OF ANTIBODIES

1. Enzymes are widely used in IHC, and are usually incubated with a ___
2. commonly used enzymes to facilitate chromogenic detection
   - ____: ____ end product (most used)
   - ____: ____ end product
3. after enzyme labeling with enzyme, it will be followed by staining with substrate or chromogen like _____
4. other methods for labeling (3)

Answer 10

1. CHROMOGEN
   2.
   - Horseradish peroxidase: brown end product
   - Alkaline phosphatase: red end product
2. diaminobenzidine
3. • fluoroscent
   • radio labels
   • colloidal metals

Question 11

DETECTION SYSTEM

______: Direct interaction between the labeled antibody and antigen

1.______ is conjugated directly to the label

Answer 11

Direct Technique
1. Primary antibody

Question 12

DETECTION SYSTEM

______: application of the unconjugated primary antibody, followed by a labeled antibody directed against the first antibody

1. most commonly used enzyme
2. unconjugated primary antibody first binds to the antigen
3. second enzyme-conjugated antibody is added to the previously described indirect technique

Answer 12

Indirect Technique

1. Horseradish peroxidase
2. Two-step Indirect Technique
3. Three-step Indirect Technique