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 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

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

moderate

Edge Artifacts from Tissue Section Drying

Higher background staining concentrated at the edges of tissue sections compared to the center, indicating tissue dehydration during the staining procedure.

💡 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

Suboptimal Primary Antibody Incubation

Inconsistent or weak staining results despite following protocol. Staining intensity varies between experiments using same antibody lot and tissue type.

💡 3 · ✓ 3