Immunocytochemistry (ICC) — Step-by-Step Protocol (Bio-Techne)

critical

Reversed Fixation and Permeabilization Order

Poor cell morphology, increased autofluorescence, or loss of intracellular antigens when permeabilization is performed before fixation.

💡 4 · ✓ 4

severe

Incorrect Permeabilization Method for Target Location

Weak or absent staining of nuclear transcription factors when using detergent-based permeabilization, or poor cytoplasmic protein detection with alcohol-based methods.

💡 4 · ✓ 4

severe

Failed Intracellular Antigen Detection After Fixation

Intracellular markers such as cytokines, transcription factors, or phosphoproteins show no signal despite proper antibody validation. Surface markers stain normally, but internal targets remain undetected indicating permeabilization issues.

💡 4 · ✓ 5