I have used tissue in 4%PFA cold (on ice) for 30 minutes if my tissue was 2-3 mm thick. If your tissue is more thick, you can leave for an hour in refrigerator. Then rinse with PBS and proceed with your next step.

For most antibodies, 4% PFA for 20min at 4C works well, but this may be dependent on your antibody. For some antibody, fixation with methanol would be more suitable. Check the spreadsheet and read several papers where they have used the same antibody, to get a consensus. If the antibody has not been tested before, make sure you have some minimal controls at least (overexpressing / knockdown cells or tissue, qPCR data, or a western blot). This article gives a nice overview of different methods https://blog.addgene.org/deep-dive-fixing-and-permeabilizing-for-immunofluorescence

What cell type are you using? I usually stain with PFA for 10-15 minutes at RT. Or in 100% Methanol for 10-15 minutes at 4C. You should consider what you are trying to stain. I.e. cell surface- PFA without detergent. Intracellular- Methanol.

4%PFA for 17-20 mins work for most of the cells.

For immunofluorescence assay (IFA), the concentration and duration of paraformaldehyde (PFA) fixation depend on the type of cells and the antigens being studied. Here’s a general guideline:

PFA Concentration

• 4% PFA in PBS is the standard concentration for most IFA applications. It provides sufficient fixation while preserving antigenicity.

Fixation Duration

• 10–15 minutes at room temperature (RT) is typical.

• Shorter times (e.g., 5–7 minutes) can be used for delicate or surface antigens to avoid masking epitopes.

• Longer times (e.g., 20–30 minutes) may be necessary for thick or dense tissues.

Procedure

1. Prepare 4% PFA fresh or thaw aliquots stored at .

2. Wash cells/tissue with PBS to remove media or debris.

3. Incubate with 4% PFA at RT for the desired time.

4. Rinse 2–3 times with PBS to remove residual PFA.

5. Permeabilize (if required) with 0.1–0.5% Triton X-100 in PBS for 5–10 minutes before antibody staining.

Tips:

• PFA fixation can alter some epitopes; if this happens, try alternatives like methanol or adjust PFA concentration/duration.

• Use freshly prepared or stabilized PFA to ensure effective cross-linking.

10 minutes PFA 4% works exellent for me in different types of cells. To mantain cells shapes you can add sucrose 5%.

4 percent PFA for about 20 minutes gives better results