Immunohistochemistry Experiment (IHC) Protocol

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

critical

Mouse-on-Mouse Cross-Reactivity in Tissue Staining

High background when using mouse primary antibodies on mouse tissue, caused by secondary antibody binding to endogenous mouse immunoglobulins and Fc receptors rather than only the primary antibody.

💡 4 · ✓ 4

severe

Over-Amplified Signal with Biotin-Based Detection Systems

Excessive background staining when using amplification techniques or biotin-based detection, either from high signal amplification or endogenous biotin binding to streptavidin/avidin.

💡 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

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

Non-Specific Binding from Elevated Incubation Temperature

Increased background staining when primary antibody incubation is performed at room temperature, as higher temperatures promote non-specific antibody-tissue interactions.

💡 4 · ✓ 4

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

Insufficient Detection System Sensitivity

Signal is weak or undetectable despite correct antibody and protocol, particularly for low-abundance targets, indicating that the detection system lacks sufficient signal amplification.

💡 3 · ✓ 4