Inadequate deparaffinization
Deparaffinize sections longer or change fresh xylene
Inactive primary antibodies
Replace with a new batch of antibodies
Antibodies do not work due to improper storage
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.
Antibody concentration was too low
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
Inadequate antibody incubation time
Increase antibody incubation time
Inadequate or improper tissue fixation
Increase duration of post fixation or try different fixatives
Tissue over fixation
Reduce the duration of post fixation. If the tissue has already been over fixed, perform an appropriate or recommended antigen retrieval procedure
Incompatible secondary and primary antibodies
Use secondary antibody that will interact with primary antibody. For example, if primary antibodies are raised from rabbits, use anti-rabbit secondary antibodies
Inactive secondary antibody
Replace with a new batch of antibodies
Inactive ABC reagents
Replace with a new batch of reagents
Defective or incompatible enzyme/substrate system
Replace with a new batch of reagents
Inadequate substrate incubation time
Increase the substrate incubation time
Incorrect mounting medium
Choose a correct mounting medium
Reagents applied in wrong order or steps omitted
Check notes or procedure used
The concentration of primary and/or secondary antibodies was too high
Reduce antibody concentration or perform a titration to determine the optimal dilution for primary and secondary antibodies
Incubation time was too long
Reduce incubation time
Incubation temperature was too high
Reduce incubation temperature
Substrate incubation time was too long
Reduce substrate incubation time
Sections dried out
Avoid sections being dried out
Inadequate washing of sections
Wash at least 3 times between steps
Tissue contains endogenous enzyme such as peroxidase or alkaline phosphatase
Block endogenous enzyme activities using 3% hydrogen peroxide (block peroxidase) in methanol or levamisole solution (block AP) prior to incubation of primary antibodies
Tissue contains endogenous biotin activity
Block endogenous biotin activity using the avidin/biotin blocking reagent prior to incubation of primary antibodies.
Non-specific binding of primary antibodies to tissue or antibody concentration was too high
Non-specific binding may be reduced by using higher dilution of primary antibodies
Non-specific binding of secondary antibodies to tissue
Treat tissue with normal serum from the same species as secondary antibodies
Diffusion of tissue antigen due to inadequate fixation
Increase duration of post fixation
Mouse antibodies used on mouse tissues
Treat tissue with MouseOnMouse blocking reagent prior to the primary antibody incubation
Sections dried out
Avoid sections being dried out
