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Immunofluorescence procedure (paraffin section) - indirect method

A. Required solutions and reagents

    1. Phosphate buffer : 0.1M 1X PBS (pH 7.4)

    2. Fixing solution: 4% paraformaldehyde solution (no methanol) 

    3. Antibody dilution : 1% BSA, 0.03% Proclin300 0.1 M PBS

    4. Antigen Repair Solution : Sodium Citrate Buffer

    5. Normal goat serum (stock solution) for closure 

    6. DAPI dye solution 

    7. Immunofluorescent anti-quenching sealant 

B. Steps

    1. 60 ° C baked 2 hours

    2. Dewaxing. Dewaxed in xylene for 15 minutes, three times

    3. Hydration. Pass through absolute ethanol, 95%, 90%, 80%, 70%, each 5 minutes, distilled water (or

Come water) 5 minutes

    4. Wash PBS  for 5 minutes, three times

    5. Antigen retrieval. Citrate buffer  (about 92-95 ° C) boiled (boiling water)

Bath) Repair for 15 minutes, naturally cool to room temperature. Wash in PBS for 5 minutes, three times

    6. Normal goat serum  is blocked, 37 ° C for 20 minutes

    7. Incubate with primary antibody at 4 ° C overnight.

    8. Rewarming for 20 minutes, washing with PBS for 5 minutes, three times

    9. Incubation by secondary antibody, 1.5 hours at 37 °C (concentration of incubation antibody, time of action through pre-experiment

Decide).

    10. Wash PBS for 10 minutes, three times

    11. DAPI counterstained nuclei, diluted 1:1000, for 5 minutes at room temperature.

    12. Wash PBS for 5 minutes, three times

    13. Anti-quenching fluorescent sealant seals, observed under the microscope.