Complete IHC Immunohistochemistry Protocol for Beginners

critical

Complete Absence of IHC Staining

Complete lack of staining observed in immunohistochemistry experiment despite using validated antibody. Tissue sections appear negative even when positive control should show signal.

💡 6 · ✓ 6

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

severe

Mouse-on-Mouse (MOM) Background

High background staining when using mouse primary antibody on mouse tissue. Secondary antibody binds endogenous mouse IgG throughout the tissue, creating non-specific signal.

💡 3 · ✓ 4

severe

Residual Reagent Background from Inadequate Washing

Elevated background signal caused by residual fixative or unbound antibodies remaining on tissue sections between procedural steps, producing false-positive staining.

💡 5 · ✓ 5

severe

Antigen Retrieval Method Not Optimized for Target

Staining results are inconsistent or suboptimal despite correct antibody and detection system, with signal strength varying significantly based on retrieval method used.

💡 4 · ✓ 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