W7- Lecture 35.3- Histology- Antibody Labeling

Question 1

Name 4 types of markers used to visualise antibodies in immunohistochemistry

Answer 1

fluorescent dye
enzyme,
radioactive element
colloidal gold

Question 2

Describe the direct method of —-

Any issues

Answer 2

Labelled monoclonal antibody (that is bound with Florescent dye )binds
with
This tissue antigen creating colour change

False positives and background interference

Question 3

Describe the indirect method

Answer 3

Primary antibody binds to tissue antigen

Secondary antibody attached to fluorescent lay labelled

They binding of the second antibody causing fluorescence

Question 4

Describe the indirect method using an enzyme

Answer 4

Primary antigen
Secondary antigen - that is biotinylated

+avidin biotinylated complex
Then enzyme substrate is added
= colour change

Question 5

Describe antigen retrieval when antigens are masked by tissue fixation (or angiogenesis )

Answer 5

To break cross linking
Tissues can be treated to LIGHTLY reverse it

E.g
Enzymatic digestion
Citric Acid
EDTA
Heat

Question 6

Endogenous tissue components may interfere with antigen detection how do we prevent this

Answer 6

Pre treat with

3 % (v/v) H2O2
0.01 % (w/v) avidin

Question 7

How do we block non specific sites to make sure only the specific antigen is recognised ?

Answer 7

10 % (v/v) normal serum

Question 8

What three pretreatments should we do before the indirect method

Answer 8

Antigen retrieval

Inhibition of endogenous tissue components

Blocking of nonspecific sites

Question 9

What controls have to be done in immunohistochemistry

Answer 9

Positive control- so make sure it triggers a response in the presence of an epitope

Diluent control- differ only

Omission of primary antibody- to make sure the antibody doesn’t bind no specifically (no indication)

Omission of secondary antibody- no colour change should be indicated

Isotype antibody- identical isotopes against different antigen (should have no effect )

Question 10

What are the advantages and disadvantages of immunohistochemistry

Answer 10

Advantages
High specificity for molecular species
Can be used for light, confocal, or electron microscopy

Disadvantages
Time consuming & expensive
Fixation can interfere with Ab binding
Reproducibility - false positives - cross reactivity
Difficult to get Abs to small molecules
Qualitative