Lecture 7: IHC Staining Patterns and Interpretation Flashcards
What is interpretation of stains based on?
Cells of interest
Previous knowledge of the antigen’s location in tissue
Knowlege of the expected staining patters (nuclear, cytoplasmic, membraneous)
Identifying background vs true staining
2 types:
Quantification or localization
What causes non-specific (background) staining?
Endogenous peroxidase Endogenous alkaline phosphatase Endogenous biotin Antigen diffusion (poor fixation) Edge artifact (tissue damage/drying) Necrosis of tissue Cross reactivity between reagents during IHC
Membraneous staining?
Stains only the cell membranes and kind of looks like a spider web version of cytoplasmic staining or like a mesh network
Nuclear staining?
Stains the nucleus, looks like a dot/ball/sphere
Cytoplasm is typically unstained
Endogenous peroxidase (background)
Found in many tissueas, esp RBCs and WBCs
This can be prevented by pre-treating the tissue with hydrogen peroxide H2O2 for 5-10 minutes prior to staining
Endogenous Alkaline phosphatase (background)
Also naturally occuring in many tissues
Can be blocke via pre-treatment with lavamisole if an alkaline phosphatase detection system is being useed in your IHC procedure
However, the AP in gastro and placental specimens is lavamisole resistant, so you should use AP detection in those tissues if you want an accurate result
What are causes of weak staining?
too dilute antibody, incorrect ab, incorrect incubation time over blocking over washing over fixation, can't be retrieved underfixed, antigens were lost insufficient antigen retrieval inadequate deparafinization incorrect mounting medium
What are causes of no staining?
Did youadd you 1 AB
did you add 2 AB
Did you add chromogen
Did you do proper antigen retrieval
was fixation adequate
Did you use the right AB on teh right tissue
DId you include proper pos and negative controls to troubeshoot
What are the results of a lack of blocking agent? (there are several)
non-specific background staining due to hydrophobic interactions that results in a uniform “muddiness” of the stain (can be prevented by using anmal blocking serum, which should be from the secondary animal, such as goat if your 2 is goat anti rabbit, to also prevent non-specific crosslinking between teh secondary and the blocking reagent
Endogenous biotin (background)
A naturally occuring acid found primarily in the liver, kidney, and brain
This background is reduced or eliminated by washing the tissue with unconjugated avidin which blocks all the natural biotin by binding to it. Then, because each avidin has 4 biotin binding sites the tissue is treated with a biotin wash to saturate the remaining free sites on avidin so it can’t bind any biotinylated primary antibodies
These blocking steps are done after the normal animal serum blcking and before application of the primary antibody (15 minute for avidin, wash, then 15 minute for biotion, and wash)
This is because antigen retrieval can expose some endogenous biotin and animal blocking serum can also conatin endogenous biotin that needs to be blocked before applying the primary antibody
necrotic tissue
doesn’t bind stains well
necrotic tissue
doesn’t bind stains well
Can be important in cancer diagnostics
specific vs non-specific background
Specific is due to antibodies binging to off target antigens while non specific is due to hydrophobic interactios and electrostatic forces often resulting in a muddy brown haze
specific vs non-specific background
Specific is due to antibodies binding to off target antigens availible due to poor fixation while non specific is due to hydrophobic interactios and electrostatic forces often resulting in a muddy brown haze
Edge artifact (background)
strong staining on edges of tissue with little to no staining in teh center of the tissue
typically due to dried out or danaged tissue during grossing, transport, or antigen retrieval
