Dear Dr. Ana Paula Sato

For double immunofluorescence staining in bovine primary fibroblasts, you can use a sequential incubation protocol using different primary antibody host species.

Protocol for double immunofluorescence staining is as follows.

1. Aspirate the cell culture medium and gently wash the coverslips twice with PBS.

2. Add a fresh solution of 4% PFA to the coverslips. Incubate for 10–15 minutes at RT.

3. Aspirate the PFA and wash the coverslips three times with PBS for 5 minutes each.

4. (For intracellular antigens only) Incubate the cells with 0.1% Triton X-100 in PBS for 10–15 minutes at RT.

5. Wash the coverslips three times with PBS for 5 minutes each.

6. Place the coverslips cell-side-up in a humidified chamber.

7. Add blocking buffer (1% BSA/PBST or 10% normal serum) and incubate for 30–60 minutes at RT.

8. Apply the first primary antibody (for instance, rabbit anti-Protein A) and incubate for 1–2 hours at RT or overnight at 4°C.

9. Incubate with a secondary antibody conjugated to a fluorophore that targets the first primary antibody (for instance, anti-rabbit conjugated to a green fluorophore) in blocking buffer for 1 hour at RT in the dark.

10. Wash the coverslip thoroughly with washing buffer to remove unbound antibodies and secondary reagents.

11. Apply the second primary antibody from a different host species (for instance, mouse anti-Protein B).

12. Incubate with a secondary antibody conjugated to a different fluorophore that targets the second primary antibody (for instance, anti-mouse conjugated to a red fluorophore).

13. Following washes, incubate with DAPI or Hoechst in PBS for 1–10 minutes to stain nuclei.

14. Rinse coverslips in distilled water, add anti-fade mounting medium to a slide, and place the coverslip cell-side-down. Seal with nail polish and image using a fluorescence microscope.

Some Tips:

A. For antibody concentration, you must standardize antibody concentration for immunofluorescence to ensure optimal signal while minimizing background noise. This involves performing a titration experiment, where you test a range of dilutions (e.g., starting around 1:100–1:1000) to find the concentration that produces clear, specific staining with low background fluorescence.

B. For blocking, the blocking buffer should contain a normal serum from the same host species as the secondary antibody, along with a detergent like Triton X-100, all in PBS. Common blocking agents are normal serum (e.g., goat serum, donkey serum) or bovine serum albumin (BSA), with concentrations typically ranging from 1-5% for serum or 1-5% for BSA. The inclusion of a detergent like Triton X-100 in the buffer is also important for permeabilizing the cells and preventing non-specific antibody binding.

C. For image setting,

a) Ensure you use a coverslip with the correct thickness, typically 0.17 mm thick.

b) Make sure the coverslip is level and all cells are in focus.

c) The filter cube must be designed for the specific fluorophore (the fluorescent dye) you are using.

d) Start with a low intensity for your lamp or laser to minimize photobleaching.

e) If the image is blurred or lacks contrast, slightly rotate the correction collar on the objective lens to find the position that offers the best image quality for your specific coverslip thickness and sample.

f) Adjust the camera's exposure time to capture enough photons without overexposing the image.

g) Increase the camera's sensitivity if the image is too dark, but avoid excessive gain, which can add noise to the image.

h) In the final check,

1) Verify that the correct metadata (scaling, acquisition parameters) are saved in your image,

2) Look at your captured image and make any final adjustments to brightness, contrast, or color balance in the software.

Best Wishes,

Malcolm Nobre

Dear Dr. Malcolm Nobre ,

Thank you for kindly sharing your protocol. Best wishes

Add.Tips:

- In sequential staining using primary antibody against the highly expressed protein as the first antibody yields better image.

- Non sequential staining can also be done, by incubating both the primary antibodies together, if time is a concern, but optimization has to be done thoroughly

- Nuclear staining and proper controls are of great help in obtaining proper images. Single antibody controls are a must.

- Seeding cell density is important. I use 5-10*10^4cells/ml.

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