Complete IHC Immunohistochemistry Protocol for Beginners

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

Weak staining / signal too faint

Positive regions show very pale staining; barely distinguishable from negative control; signal appears dotted or discontinuous.

💡 5 · ✓ 5

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

Weak or absent signal from inadequate antibody concentration or incubation

No detectable staining despite proper protocol execution. Insufficient antibody-antigen binding due to suboptimal concentration or incubation conditions.

💡 4 · ✓ 5

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

moderate

Edge effect / uneven staining across the slide

Slide edges show darker staining than center, or one half stains more than the other; reproducibility between fields is poor.

💡 4 · ✓ 4

moderate

Loss of membrane protein staining after permeabilization

No staining is detected when targeting membrane proteins in IHC. Signal is absent despite antibody validation, suggesting structural damage to membrane epitopes.

💡 3 · ✓ 4