Weak or No Staining
| Sources | Solutions |
| 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 postfixation or try different fixatives |
| Tissue overfixation | Reduce the duration of postfixation. If the tissue has already been overfixed, 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 antibody |
| 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 |
Overstaining
| Sources | Solutions |
| 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 |
High Background
| Sources | Solutions |
| 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. |
| Secondary antibodies cross react with similar species of tissue, i.e. rabbit anti-rat IgG may cross react with mouse tissue. | Use pre-adsorbed 2nd antibody, i.e. use rabbit anti-rat IgG, mouse adsorbed, on mouse tissue, or use rabbit anti-mouse IgG, rat adsorbed, on rat tissue. |
| Diffusion of tissue antigen due to inadequate fixation | Increase duration of postfixation |
| 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 |