Weak/No Specific Signal

1. Antibody inefficiency (expired, mismatched epitope);

2. Inadequate antigen retrieval (insufficient epitope exposure);

3. Over-blocking or excessive washing;

 4. Incompatible fluorochrome/chromogen

1. Validate antibody specificity and optimize dilution ratio (1:50–1:200);

2. Select target-adapted retrieval method (citrate buffer/EDTA, 95–100℃ for 15–20min);

3. Adjust blocking time (1–2h at room temperature) and washing cycles (3×5min with PBS-T);

4. Choose compatible labels (e.g., fluorochromes with non-overlapping emission spectra for IF-based mIHC)

High Non-Specific Background

1. Excessive antibody concentration;

2. Inadequate blocking (improper blocking buffer selection);

3. Cross-reactivity between primary and secondary antibodies;

4. Unoptimized signal amplification steps

1. Reduce antibody concentration (e.g., adjust from 1:100 to 1:200);

2. Block with species-matched serum or 5% BSA;

3. Set isotype controls and use monospecific primary antibodies plus cross-adsorbed secondary antibodies;

4. Optimize amplification steps (e.g., shorten tyramide signal amplification (TSA) incubation time)

Signal Overlap/Cross-Talk

1. Overlapping fluorochrome emission spectra;

2. Incomplete antibody stripping (during sequential staining);

3. Cross-reactivity of detection reagents

1. Select fluorochromes with non-overlapping emission spectra (e.g., Alexa Fluor 488/594/647);

3. Optimize stripping protocol (e.g., treatment with glycine-HCl buffer pH 2.2 for 10–15min);

3. Add reagent-specific blocking steps and verify detection system compatibility

Uneven Staining

1. Uneven antibody incubation (reagent pooling);

2. Inconsistent tissue section thickness;

3. Inhomogeneous antigen retrieval;

4. Precipitates in antibodies/reagents

1. Incubate on a shaker (50–100rpm) to ensure full sample coverage by reagents;

2. Standardize section thickness (3–5μm);

3. Ensure uniform heating during retrieval;

4. Centrifuge reagents (10,000rpm for 5min) to remove precipitates before use

Signal Quenching

1. Poor fluorochrome stability;

2. Failure to use anti-fade mounting medium;

3. Prolonged light exposure;

4. Excessively high ambient temperature

1. Choose photostable fluorochromes (e.g., Alexa Fluor, DyLight series);

2. Mount sections with anti-fade mounting medium containing DAPI;

3. Reduce excitation light exposure time and use fast-capture imaging systems;

4. Perform staining and imaging at 4℃ or on ice whenever possible

Tissue Damage

1. Harsh antigen retrieval conditions (overheating/prolonged duration);

2. Excessively high detergent concentration (e.g., Triton X-100);

3. Mechanical damage during washing/stripping;

4. Incompatible fixative

1. Optimize retrieval conditions (lower temperature/shorten duration);

2. Reduce detergent concentration (0.1% Triton X-100);

3. Wash gently to avoid vigorous shaking;

 4. Fix with 4% paraformaldehyde at room temperature for 15–20min, avoiding over-crosslinking fixation

Inconsistent Signal Intensity Between Runs

1. Antibody batch differences;

2. Non-uniform reagent concentrations;

3. Uncontrolled incubation temperature/duration;

4. Inconsistent tissue processing workflows (fixation/storage)

1. Use antibodies from the same batch, aliquot and store at -80℃;

2. Standardize reagent preparation protocols;

3. Use temperature-controlled incubators and strictly control incubation time;

 4. Standardize tissue fixation (immediate fixation after collection) and storage (store paraffin sections at -20℃)