LECTURE - Stain Troubleshooting + Immunohistochemistry

Question 1

general approach to troubleshooting

Answer 1

• look at slide under microscope and compare to ideal
• identify incorrect step
• remove coverslip; soak in xylene
• take back down to water
• restart stain at that step (that you thought was deficient)
• continue to end
• coverslip
• review again

Question 2

what do you do when troubleshooting and two steps are equally likely the cause of the poor stain?

Answer 2

choose one and experiment

Question 3

imunohistochemistry

Answer 3

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

Question 4

when is IHC performed?

Answer 4

after a pathologist has reviewed the routine H&E and/o special stains

anticipatory: patient already has diagnosis or relevant clinical history

Question 5

rationale for IHC

Answer 5

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

specific and sensitive for detecting pathological antigens; important in cancers

Question 6

polyclonal antibodies

Answer 6

Ag injected into an animal and it makes Abs => harvest serum of that animal => extract variety of antibodies

Question 7

monoclonal Abs

Answer 7

inject Ag to animal => harvest spleen cells where Abs are produced (spitting out one type of Ab) => hybridize with myeloma cells => hybridoma = spits out one type of Ab

Question 8

primary vs secondary antibody

Answer 8

The primary antibody detects the antigen in the specimen, but the secondary antibody can be designed to have a fluorophore or enzyme complex attached to it for the purposes of visualization

Question 9

polyclonal vs monoclonal antibodies

Answer 9

• polyclonal is less expensive (we dilute monoclonal bc very expensive)
• polyclonal = mixed population of Abs; monoclonal is single Ab
• polyclonal binds to multiple areas of target antigen whereas monoclonal binds to specific area only

Question 10

when primary antibody is labeled

Answer 10

direct IHC

Question 11

this binds specifically to antigen

Answer 11

primary antibody

Question 12

this binds to primary antibody

Answer 12

secondary antibody

Question 13

when secondary antibody is labelled

Answer 13

indirect IHC; signal is amplified

Question 14

label and detection in IHCC

Answer 14

• bound to Ab or third layer = enzymes slike HRP or polymers
• chromogen added (allows us to see label) = DAB (brown) or AP (red)
• *HRP reacts with the above to produce colour**
• counterstain = hematoxylin is most frequent (blue)

Question 15

3rd layers in IHC

Answer 15

are additional specific layers to increase sensitivity and reduce amount of primary and secondary antibody needed

Question 16

examples of third layers

Answer 16

• PAP = peroxidase anti-peroxidase; 3rd layer ab binding to second layer
• ABS = avidin-biotin complex; uses biotinylated secondary Ab; 3rd layer is avidin-peroxidase complex
• LSAB = labeled streptavidin-biotin = similar to ABS with substitute

Question 17

frozen tissue sections are used for

Answer 17

immunofluorescence

• fluorophore-linked secondary or primary abs instead of enzyme; use fluorescent microscope!
• can use multiple labels with the same tissue by using different coloured fluorophores

BUT fluorescence doesn’t last long….

Question 18

Disadvantages of immunofluorescence IHC

Answer 18

• fluorescence doesn’t last long
• many steps
• different antibodies need different protocols