Hi!

There could be many reasons.

Most importantly, not all antibodies still recognize their epitope after fixation. For that you could try to fix with PFA (e.g. 4% in PBS). Also check whether you can find an antibody that has been tested for histology, not only for Western Blotting or FACS.

Second, your FXR is a nuclear receptor, correct? Did you permeabilize the cells in any way? That could be neccessary. There are different options. If you have very thin sections, e.g. 5-10 micron sections that often adding some Triton or tween to the washing buffer does the trick. Fo thicker sections, one can permeabilize the tissue before antibody staining with the eBioscience FoxP3 staining buffer kit.

Third, you have a lot of unspecific AF488 signal, indicating that sections should be probably washed longer...? Or amount of secondary antibody reduced.

Hi!

There could be many reasons.

Most importantly, not all antibodies still recognize their epitope after fixation. For that you could try to fix with PFA (e.g. 4% in PBS). Also check whether you can find an antibody that has been tested for histology, not only for Western Blotting or FACS.

Second, your FXR is a nuclear receptor, correct? Did you permeabilize the cells in any way? That could be neccessary. There are different options. If you have very thin sections, e.g. 5-10 micron sections that often adding some Triton or tween to the washing buffer does the trick. Fo thicker sections, one can permeabilize the tissue before antibody staining with the eBioscience FoxP3 staining buffer kit.

Third, you have a lot of unspecific AF488 signal, indicating that sections should be probably washed longer...? Or amount of secondary antibody reduced.

Nuclear staining would work always all the times since it’s the easies. 4% PFA/PBS for 20 min at RT is a good way to fix the cells. Then wash with 1x TBS-T to wash (you can google the recipe, the most common washing buffer). Then you have to block your cells, 3% BSA/2x TBST would work for 1 hr at RT. Then add 1st Ab diluted in 3% BSA/ 1xTBST according to recommendation for overnight at 4degree. Make sure it cover up your cell/ or tissue. Then wash, add 2nd Ab, mostly at 1:1000 DF for 2-3 hr at RT. Wash, nuclear stain, mount with correct mounting media with cover glass. Wait until dry up, visualize.

From your result, I see lots of green background. I think your blocking step might not enough since you didn’t mention this step in your experiment.

You can try to adjust green channel with your negative control, which shouldn’t have any 1st antibody added sample.

As detailed in the method you followed, you did not block the cells which is usually followed to prevent non-specific binding, reduce background interference and improve the signal-to-noise ratio. Secondly, as Morbe mentioned, not all antibodies recognize their epitope after fixation. So, better go for fixing step at the end.

In simple, after removing the culture medium, give a quick wash and block the cells with 5% (w/v) BSA in PBS (pH 7.4) for 30min on ice. If you are using indirect staining, it is recommended to add serum (5%) ideally derived from the same species in which the secondary antibody is raised. If the cells which you are staining express Fc receptor, then it would be necessary to add Fc block (1:500) in the blocking buffer. Macrophages do express Fc receptors. Following washes with PBS, incubate the cells with antibodies (either primary conjugated in direct staining or primary and secondary as in indirect staining). Then fix with 1% w/v formaldehyde in PBS. Of course, nuclei staining is by default for which you can choose a separate stain as you did or use a mounting medium with DAPI.

Importantly, washing to remove unbound antibody is really needed.

Urs Mörbe thank you so much for your suggestions! I read online that permeabilization is not necessary when methanol is used for fixation, because this already leads to permeabilization. This is why I choose methanol, would you agree with this?

Perhaps something went wrong with the washing indeed or the dilution. I washed with PBS now three times for 5 minutes. The secondary antibody I used was Alexa Fluor® 488 Goat Anti-Mouse (IgG) secondary antibody (ab150113) | Abcam at a dilution of 1:500, yet perhaps I should dilute it more then?

Enkhtuya Radnaa thank you so much for your suggestions! I forgot to mention the blocking step indeed, yet I did include this. I incubated my cells in 1% BSA in PBS, for 30 minutes at room temperature. Both the primary and secondary antibody were incubated with the sample for 1 hour. Do you think the incubation time could make a big difference for the results?