Hi Pawel, not so easy to troubleshoot with little detail and almost no signal at all to start with! Anyway I'll try with a few suggestions:

1. Paraffin embedded tissue is the most difficult for IF, so I would leave it aside at the beginning, until you get something from the frozen one.

2. Fixation, better short, not more than 10 min in 4% formaldehyde in PBS at room temp. Some Abs prefer even 2% formaldehyde.

3. I would do a 1 h treatment with 50 mM NH4Cl at room temp. to quench carboxylic-derived fluorescence (from formaldehyde and other endogenous molecules)

4. Blocking might be too strong, start with 2% NGS (if your secondaries are raised in goat, otherwise horse or donkey serums are also fine) and 2% BSA. If you have too much background, rise to 5% NGS (with this conc. you can leave out the BSA, there is enough in the serum)

5. I would permeabilize the frozen sections. 0.2-0.3% triton will be fine. I would use it in all steps, blocking, primary incubation, washes, etc... You have a section probably around 10-12 um thick, if I remember correctly, adipocytes are pretty small and in a tissue there is always quite a lot of extracellular matrix, so it is likely that lipids will not escape from the section, will just form local micelles; if you remove the triton they can reclose, reform some kind of membranes, hindering the access of the Abs.

6. You might want to include 1-2% DMSO in the blocking and the primary Ab solution, it helps accessing difficult proteins (I'm not at all expert in adipocytes and have no clue where the proteins you are studying are localized in the cell, but if they are embedded in protein matrixes they will be difficult to access).

7. Primary antibody dilution: I would stay with the low dilution (so keep them concentrated). If you get too much signal or too much background you can try diluting, hoping the signal stays and the background falls...

8. Finally incubation conditions for the primaries: I would try in parallel 3 conditions, 30-60 min at 37 °C, O/N at room temp., 48 hrs at 4 °C (assuming you tried the standard O/N at 4 °C... if you only tried 1 to few hrs at r.t. then add the O/N at 4 °C to your trials!)

9. Secondary concentration is normally not an issue, typically between 1:400 to 1:1000 is always fine. Focus on blocking, permeabilizing, and primary incubation!

Good luck!

 Dear Pietro,

thank you for your insight! I'll try to follow your guidelines and let you know how it goes.

p.s. do you think I should use TRITON and DMSO together during blocking?

Hi Pawel, it's one of the possibilities. We use it regularly in brain floating sections, which are 20 um thick, mostly to access pre- or post-synaptic compartments. In cryosections we use it rarely, because they are thinner, but in certain cases we can't avoid it. Honestly, I have no experience with adipose tissue, except that I imagine you will have quite some fat in the cells. DMSO is a very versatile chemical, solubilizing both polar and non-polar substances and miscible with both water and organic solvents. In protein matrixes (highly hydrophobic) and in fat it will create holes, pores, increasing accessibility to water soluble Abs. If you don't get any staining in standard conditions, before giving up, try to add it to your blocking, together with Triton. You can also keep it with the primary and secondary antibodies if you use a 1-2%. In the blocking you can go up to 4%.

Good luck!

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