No/Weak Fluorescent Signal

1. Insufficient antigen retrieval or improper method;

2. Over-fixation damaging epitopes;

3. Fluorescence quenching (expired reagents, non-dark operation);

4. Inadequate incubation time/temperature

5.Primary/secondary antibody inactivation, mismatched species, or over-dilution;

1. Select appropriate retrieval (citrate buffer heat retrieval for proteins, trypsin digestion for nucleic acids);

2. Optimize fixation (4% PFA for 15-20min, avoid high temperature/prolonged fixation);

3. Operate in dark, use fresh fluorescent reagents;

4. Incubate primary antibody at 4℃ overnight, secondary at RT for 1-2h

5. Verify antibody validity, adjust dilution (primary: 1:100-1:500, secondary: 1:200-1:1000), store in aliquots;

High Non-Specific Background (Overall Glow)

1. Excessive antibody concentration;

2. Inadequate blocking (improper buffer or short time);

3. Incomplete washing;

4. Secondary antibody cross-reactivity;

5. Sample autofluorescence (e.g., collagen, pigment cells)

1. Reduce antibody concentration (e.g., primary 1:200 → 1:500);

2. Block with 5% BSA/serum for 1-2h at RT (match secondary antibody species);

3. Wash with PBS-T (0.1% Tween-20) 3-4 times (5-10min each);

4. Use monoclonal primary antibody and species-matched secondary antibody;

5. Use quencher or long-wavelength fluorochrome for autofluorescent samples

Uneven Fluorescent Signal (Spotted/Local High Signal)

1. Uneven antibody distribution (insufficient shaking during incubation);

2. Precipitates in antibodies/blocking buffer;

3. Bubbles or uneven sample mounting;

4. Insufficient reagent volume to cover sample

1. Incubate on shaker (50-100rpm);

2. Centrifuge reagents (10000rpm, 5min) to remove precipitates;

3. Avoid bubbles during mounting, ensure tight sample-slide adhesion;

 4. Use ≥100μL reagent per slide to fully cover sample

Rapid Fluorescence Quenching

1. Unstable fluorochrome (e.g., FITC);

2. No anti-fade mounting medium;

3. Prolonged excitation light exposure

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

3. Mount with anti-fade medium after staining;

3. Shorten excitation light exposure time, avoid continuous irradiation

Damaged Cell Morphology

1. Excessive fixative concentration or prolonged fixation;

2. Severe antigen retrieval conditions (e.g., overheating);

3. Improper buffer osmolarity during washing;

4. Mechanical damage (e.g., vigorous pipetting)

1. Use 4% PFA, shorten fixation to 15-20min;

2. Optimize retrieval (95-100℃ for 10-15min);

3. Use isotonic PBS for washing;

4. Operate gently, avoid vigorous pipetting/scraping

Abnormal Specific Fluorescence Localization

1. Epitope masking (e.g., protein folding);

2. Incorrect primary antibody recognition site;

3. Delayed fixation causing antigen translocation;

4. Over-permeabilization (e.g., high Triton X-100 concentration for membrane proteins)

1. Optimize retrieval or use detergent to expose epitopes;

2. Confirm primary antibody recognition site matches target protein localization;

3. Fix samples immediately after collection;

 4. Use 0.1%-0.2% Triton X-100 for permeabilization, omit for membrane protein detection