Little or no label |
Antigen not exposed |
Use permeabilizing agent, i.e. saponin |
Fixation disrupts/alters antigen |
Titrate or change fixatives |
Primary or secondary concentration too low or not specific |
Titrate antibodies, try alternative antibodies |
Steric hindrance of large antibodies |
Use smaller probes |
Loss of structure |
Find alternative fixation additives, i.e. alcian blue or ruthenium red |
Cross-reactivity |
Change ab species |
Excessive label on specimen |
Concentration of primary and/or secondary ab too high |
Dilute antibody concentration |
Increase NaCl concentration |
Non- specific binding to specimen |
Change blocking buffer |
Quench aldehydes by adding 0.1% glycine |
Excessive label on background substrate |
Concentration of primary or secondary ab too high |
Titrate ab concentration |
Add or change blocking agent |
Poor elemental contrast in microscope |
Gold size too small |
Use larger gold size or enhance with silver or gold |
Sub-optimal microscope settings |
Use larger aperture, greater voltage, bigger spot size, shorter working distance |
Coating or OTOTO masked gold |
Apply lighter coat of low atomic number metal and/or reduce OTOTO incubation time. |
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