Table 2.

Troubleshooting guide for immune-labeling

Problem	Possible Cause	Possible Remedy
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.