Immunofluorescence experiment checklist

Specimen

• Sample identifier, biological replicate, collection date
• Cell line, passage, density, treatment, or tissue source and processing
• Imaging surface, slide type, section thickness, and storage history

Staining

• Fixation condition and rationale
• Permeabilization or retrieval condition and rationale
• Blocking reagent, time, temperature, and volume
• Primary and secondary antibody identifiers, lots, dilutions, and incubation conditions
• Wash composition, count, duration, volume, and agitation
• Counterstain and mounting medium

Controls and imaging

• Unstained, secondary-only, positive, negative, and single-color controls as applicable
• Microscope, objective, immersion medium, channels, exposure, gain, binning, and z-step
• Raw files retained and processing steps documented
• Comparison groups acquired with compatible settings and no saturation