Weak or absent staining |
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Antigen levels are too low |
Prolong incubation time of primary antibody |
Use a higher sensitivity staining system |
Incomplete fixation |
Prevent under (> 30 min) or overfixation (> 48 hr) |
Use of inappropriate fixative |
Check manufacturer’s specifications regarding recommended fixative |
Insufficient dehydration |
Operating regular reagent changes (i.e., alcohol) |
Paraffin too hot |
Monitor temperature of paraffin (< 60°C) |
Embedding and dewaxing at high oven temperature |
Oven temperature not to exceed 60°C |
Heating for antigen retrieval |
Optimize antigen retrieval time |
Reagents not working, reagents in wrong order |
Monitor expiration dates, storage parameters, and pH |
Antibody too dilute, improper antibody dilution |
Determine correct concentration |
Check incubation time and temperature |
Partial drying out of tissue during processing |
Immerse tissue immediately in fixative |
Use a huminity or moist chamber during incubation steps |
Avoid evaporation with humidity chamber |
Chromogen not working, incorrect preparation of chromogen |
Add chromogen to labeling sdution |
Monitor for change in color |
Background or artifactual staining |
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Excessive incubation |
Reduce incubation time |
Necrotic or otherwise damaged tissue |
Avoid sampling of necrotic areas |
Make sure tissue is properly fixed |
Antigen diffusion before fixation leading to specific background |
Avoid delays in fixation |
Thick preparation |
Cut sections at 4 to 6 mm |
Inappropriately concentrated antibody |
Check titration and concentration |
Decrease temperature of reaction |
Presence of chromogen or undissolved counterstain deposits |
Filter the chromogen or counterstain |
Insure that chromogen is completely dissolved |
Incomplete inadequate rinsing of slides |
Follow protocol for proper slide rinsing |
Mildly rinse slide with wash buffer bottle and place in wash bath in 5 min |
Endogenous pigments |
Check the negative control for the presence of these pigments |
Use a chromogen of contrasting color |