Troubleshooting
Start from what you see in the image.
Check the relevant controls, staining pattern, channel behavior, and acquisition settings before changing the protocol. Change one factor at a time so the result is interpretable.
Signal
No or weak signal
Check specimen presence, positive controls, antibody access, staining reagents, and imaging.
Open guide → SpecificityHigh background
Use unstained and detection controls to separate specimen background, antibody background, and overexposure.
Open guide → SpecimenAutofluorescence
Measure unstained background by channel before choosing quenching or alternative fluorophores.
Open guide → Spatial patternUneven staining
Check reagent coverage, drying, bubbles, illumination, focus, and section integrity.
Open guide →