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Weak or No Staining

Deparaffinize sections longer or change fresh xylene

Aliquot antibodies into smaller volumes and store in freezer (-20 to -70 C) and avoid repeated freeze and thaw cycles. Or store antibodies according to manufacture's instructions.
Increase the concentration of primary and/or secondary antibodies. Or run a serial dilution test to determine the optimal dilution that gives the best signal to noise ratio
Increase antibody incubation time
Increase duration of post fixation or try different fixatives
Reduce the duration of post fixation. If the tissue has already been over fixed, perform an appropriate or recommended antigen retrieval procedure
Use secondary antibody that will interact with primary antibody. For example, if primary antibodies are raised from rabbits, use anti-rabbit secondary antibodies
Replace with a new batch of antibodies
Replace with a new batch of reagents
Replace with a new batch of reagents
Increase the substrate incubation time
Choose a correct mounting medium
Check notes or procedure used


Overstaining

Reduce antibody concentration or perform a titration to determine the optimal dilution for primary and secondary antibodies
Reduce incubation time
Reduce incubation temperature
Reduce substrate incubation time
Avoid sections being dried out


High Background

Wash at least 3 times between steps
Block endogenous enzyme activities using 3% hydrogen peroxide (block peroxidase) in methanol or levamisole solution (block AP) prior to incubation of primary antibodies
Block endogenous biotin activity using the avidin/biotin blocking reagent prior to incubation of primary antibodies.
Non-specific binding may be reduced by using higher dilution of primary antibodies
Treat tissue with normal serum from the same species as secondary antibodies
Increase duration of post fixation
Treat tissue with MouseOnMouse blocking reagent prior to the primary antibody incubation
Avoid sections being dried out