Complete IHC Immunohistochemistry Protocol for Beginners

critical

False-Positive Signal from Endogenous Enzyme Activity

High background signal observed when using enzyme-conjugated antibodies (HRP or AP), caused by endogenous alkaline phosphatase or peroxidase activity in tissue generating false-positive results.

💡 4 · ✓ 4

severe

Tissue detachment / sections fall off the slide

Tissue falls off during staining; sections become broken or missing; results cannot be interpreted.

💡 4 · ✓ 4

severe

High Background from Insufficient Non-Specific Blocking

Diffuse background staining observed throughout tissue sections in IHC experiments, indicating inadequate blocking of non-specific antibody binding sites.

💡 4 · ✓ 4

severe

High Background from Excessive Antibody Concentrations

Elevated background staining throughout tissue sections due to excessively high concentrations of primary or secondary antibodies leading to non-specific binding.

💡 4 · ✓ 5

moderate

Nuclear protein not detected due to insufficient permeabilization

No staining observed when targeting nuclear proteins. Antibody cannot access nuclear compartment despite proper antibody concentration and incubation time.

💡 3 · ✓ 4

moderate

Inadequate Washing Steps Causing Poor Contrast

High background persists with poor contrast between positive signal and background despite other optimization steps, making interpretation difficult.

💡 4 · ✓ 4