Immunofluorescence Protocol

Immunofluorescence Protocol

General protocol for immunofluorescence. Please ensure you understand all steps and safety guidelines well before you proceed.


1. Primary and secondary antibodies

2. Phosphate-buffered saline (PBS)

3. Blocking solution

4. PFA fixative

5. Detergent (Triton X-100)

6. Glass slides & cover slip


1. Fixation:

  — Grow cells on a coverslip properly.

   — Fix the cells using a suitable fixative like paraformaldehyde (4% PFA for 15 minutes at room temperature).

  — Be careful while handling PFA, as it is harmful.

2. Permeabilization:

  — After fixation, rinse cells/ tissue three times with PBS.

   — Permeabilize the cells with 0.1% Triton X-100 in PBS for 10 minutes.

3. Blocking:

   — Rinse with PBS and block with a blocking solution (like 1% BSA in PBS or 5% goat serum, as needed) for 1 hour at room temperature.

4. Primary Antibody Incubation:

   — Incubate with a primary antibody for overnight at 4 Degrees Celsius or for 1-2 hours at room temperature.

5. Washing:

   — Post incubation, wash the cells three times, five minutes each wash with PBS.

6. Secondary Antibody Incubation:

   — Incubate with secondary antibodies (usually fluorescently labelled – FITC, TRITC, Cy5, etc.) for 1-2 hours in the dark at room temperature.

7. Washing:

   —  Rinse the cells three times with PBS for five minutes each.

8. Mounting:

   — Mount the slides using a mounting medium.

9. Microscopic Examination:

   — Perform microscopic examination under suitable conditions.

In addition to this general protocol, you must consider that specific tissues, cells, or experiments may require modifications or additional steps for optimal results.

Remember: Always follow your lab’s safety regulations, especially when working with potentially hazardous reagents or equipment like fixatives or fluorescence microscopes.

Note: This procedure requires practice and knowledge in cell culture and fluorescence microscopy. If you need assistance, consult with a mentor or someone with experience in the immunofluorescence technique.

Dr AF Saeed

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