immunohistochemistry

Question 1

what is immunohistochemistry

Answer 1

method for detecting specific antigens on cells using the antigen/antibody reaction

Question 2

when is IHC performed

Answer 2

• after routine stains are views
• patient already has diagnosis or relevant clinical history

Question 3

what does IHC used for

Answer 3

• detects pathological antigens
• can assign lineage to cells/tumors
• determine stage and grade of cancers
• personalized/ precision medicine treatments
• allows us to see distribution and localization of antigens within the tissue

Question 4

what does IHC detect

Answer 4

many of different antigens

Question 5

what antibodies can be detected

Answer 5

• polyclonal vs monoclonal
• primary vs secondary

Question 6

polyclonal is…

Answer 6

injecting antigen to animal to raise an immune response to harvest the antibody
- the Ab is directed against different parts of antigen

Question 7

monoclonal is..

Answer 7

specific to single part of antigen which raises the immune response
- spleen cells is where the antibody is used in

Question 8

components of polyclonal

Answer 8

• less expensive
• mixed population of antibodies
• binds to multiple areas of target antigen
• cause cross reactivity less specific

Question 9

monoclonal components

Answer 9

• very expensive
• single antibody
• binds to specific areas of antigen only

Question 10

primary

Answer 10

binds specifically to antigen
- primary is labelled = direct IHC

Question 11

secondary

Answer 11

binds to primary antibody
- when secondary is labelled = indirect IHC
- signal is amplified

Question 12

how is antigen labelled and detected

Answer 12

• bound to antibody or third layer (enzymes and polymers)
• chromogen added (DAB brown AP red)
• counterstain

Question 13

why are third layers used

Answer 13

addition specific layers to increase sensitivity and reduce amount of primary and secondary antibody

Question 14

examples of third layer

Answer 14

PAP
ABC
LSAB
polymer method

Question 15

PAP

Answer 15

peroxidase anti peroxidase
- third layer antibody binding to second layer

Question 16

ABC

Answer 16

avidin-biotin complex (3rd layer)
- uses biotinylated secondary antibody

Question 17

LSAB

Answer 17

labeled streptavidin biotin
- similar to ABC but the complex is not pre assembled

Question 18

polymer method

Answer 18

dextran polymer can bind up to 70 enzyme molecules along with primary or secondary antibody

Question 19

what are frozen tissue section used for

Answer 19

• used for immunofluorescence
• fluorophore- linked secondary or primary antibody
• can use multiple labels with the same tissue by using different colour chromophores

Question 20

true or false: different antibodies need different protocols

Answer 20

true

Question 21

there can be no delays in…

Answer 21

fixation

Question 22

routine formalin..

Answer 22

cross links mask antigens

Question 23

epitope retrieval steps

Answer 23

• heat induces epitope retrieval
• enzyme induces epitope retrieval

Question 24

microtomy

Answer 24

needs to be skilled

Question 25

this step needs to be complete

Answer 25

paraffin removal

Question 26

quenching/ blocking steps

Answer 26

incubate tissue with solutions that bind to non specific sites

Question 27

quenching/ blocking steps help..

Answer 27

reduce background staining reduce false positive reactions

Question 28

primary antibodies must be

Answer 28

optimized

Question 29

what are controls that are used

Answer 29

positive controls
biologically negative controls
reagent negative controls
must be able to contrast with special stain control

Question 30

positive controls

Answer 30

specimens containing the target molecule

Question 31

biologically negative controls

Answer 31

specimens that do not contain the target molecule

Question 32

reagent negative controls

Answer 32

omit the primary antibody
- assess background staining

Question 33

contrasting with special stain control

Answer 33

single section of biologically positive tissue on the same slide as the patient