Immunofluorescence Protocol (FFPE)

Immunofluorescence Protocol (FFPE)

A general protocol for performing immunofluorescence on formalin-fixed paraffin-embedded (FFPE) samples.


1. FFPE tissue sections/slides

2. Primary and secondary antibodies

3. Xylene

4. Ethanol in various concentrations

5. Citrate buffer (pH 6.0) or similar antigen retrieval solution

6. Blocking solution

7. Phosphate-buffered saline (PBS)


9. Mounting medium


1. Deparaffinizing:

   — Immerse slides in xylene for two successive 5 minutes to remove paraffin.

   — Rehydrate cells in a graded alcohol series (100%, 95%, 70%, and 50%) for 3 minutes each and then in distilled water.

2. Antigen Retrieval:

   — Heat slides in citrate buffer (pH 6.0) or another antigen retrieval solution in a microwave or pressure cooker.

   — Allow to cool at room temperature for 20-30 minutes.

3. Blocking:

   — Incubate slides with a blocking solution (like 5% goat serum in PBS) for 1 hour at room temperature to reduce nonspecific antibody binding.

4. Primary Antibody Incubation:

   — Apply your primary antibody to the tissue section and incubate overnight at 4°C in a humidified chamber.

5. Washing:

   — Rinse the slides with PBS three times, for 5 minutes each wash.

6. Secondary Antibody Incubation:

   — Apply fluorescently labelled secondary antibody to the tissue section and incubate for 1-2 hours at room temperature in the dark.

   — Wash the slides with PBS again.

7. Staining:

   — Some protocols may include counterstaining with DAPI to stain the nuclei, which takes about 5 minutes.

8. Mounting:

   — Coverslip the slides using an aqueous mounting medium that’s suitable for fluorescence microscopy.

9. Microscopic Examination:

   — Image your slides with a fluorescence microscope.

Note: Your specific tissue, antigen, and antibody may require adjustments to the above protocol, such as different antigen retrieval methods or incubation times. Always follow any specific instructions from the antibody manufacturer or other trusted sources. And please make sure that you follow all your lab safety rules.

Dr AF Saeed

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