Immunohistochemistry: immunostainning

Question 1

Principal immunotechniques

Answer 1

Enzyme based detection methods

Fluorescence based detection methods

Question 2

What does imumunodetection methods based on?

Answer 2

In the interaction between antibodies and molecular targets in tissues called antigens

Question 3

Epitope

Answer 3

Small section of antigen 8 to 15 sequence of aminoacids recognized by antibody

Question 4

Molecule which induces production of antibodies

Answer 4

Antigen

Question 5

Types of antibody production

Answer 5

Monoclonal and polyclonal

Question 6

Stages of monoclonal antibody production

Answer 6

Injection of antigen to animal normally mouse (immunization)

Isolate B cells from spleen (Antibody forming cells)

B cell fusion with tumor cell resulting in hybridoma that grows indefinitely

Isolate cells of hybridoma that secrete antibody that interact with epitope of antigen

Clonal expansion

Question 7

Characteristics of polyclonal antibody production

Answer 7

Injection of antigen to animal generally a rabbit (immunization)

B cells produce antibody found in serum of the animal

Different B cells binds to different epitopes of the same protein

Variation between production batches

Question 8

Types of antibodies

Answer 8

Immunoglobulin family

IgG, IgM, IgA, , IgD, IgE

Question 9

Most commons Ig

Answer 9

IgG and IgM

Question 10

Number of chains in immunoglobulins

Answer 10

4 chains , 2 heavy and 2 light

Question 11

Types of immunohistochemistry protocols

Answer 11

Direct and indirect

Question 12

BrdU staining use

Answer 12

To assess neural stem cell proliferation in certain tissue

Question 13

Sections of immunostaining sections

Answer 13

1. Incorporation of positive and negative controls
2. Antigen retrieval
3. Blocking of non specific binding

Question 14

Describe positive control incorporation

Answer 14

Use a different tissue that express same protein of study to compare

Question 15

Describe negative control incorporation

Answer 15

Omit primary antibody from normal serum from animal that the secondary antibody was raised in instead

Question 16

What is antigen unmasking or retrieval?

Answer 16

Any technique where the masking of an epitope is reversed so that the antibody can again bind to it

Question 17

Name techniques of antigen retrieval

Answer 17

Heat induced epitope retrieval (HIER)

Protease induced epitope retrieval

Question 18

Why we block for non-specific binding?

Answer 18

Because antibodies can bind to no specific antigens causing false positive signal

Question 19

How we block for non - specific binding?

Answer 19

We use proteins from the serum of an animal which compete with antibodies for non specific binding

Question 20

Characteristics of direct method of immunohistochemistry

Answer 20

Primary antibody linked direct to a report molecule

Only suitable for highly expressed proteins