Advice for preventing quick loss of fluorescence intensity?
Hi everyone,
I'm having problems setting up a new staining in PBMCs and I don't know what else I can troubleshoot, so I would appreciate any imput.
Problem: significant loss of fluorescent signal in mounted coverslips within less than 24h.
We think the problem is in sample preparation. This is the first time doing microscooy in this lab and in PBMCs, but I have done microscopy before with other cell lines in other labs, so I thought I could just reuse those protocols. Please don't assume anything, we might be messing up in something very obvious and small.
Goal is to stain 2 intracellular proteins to measure their precence (and ideally at some point do some colocalization, but let's start with visualizing them..). Initial stainings worked, although signal needed to be improved. Sometimes I couldn't image on the same day that the slides were ready and then when I tried to image the next day I would have lower intensity than previous tests and I would be very confused.
Basic protocol:
Since PBMCs are non-adherent, we have tried puting them in fibronectin(or Poly-D-lys)-coated coverslips either at the beginning or at the end of the staining protocol. We noticed better cell numbers doing it after, so we are doing stainings as we would for flow cytometry in a 96-well plate. Initial stainings show no difference between the approaches, but please let me know if anyone thinks this might be not helping.
(Antibody mix and washing steps with PBS 0.5% BSA)
- Take PBMCs from culture, wash 3x
- Fix 4% PFA, we have tested both 30min 4°C and 10 min RT. Wash 3x
- Permeabilize 1% triton + 5% BSA, 10min RT. Wash 3x
- Block 5% BSA. Wash 3x
- Stain with primary antibodies (one mouse, one rabbit). 4°C, tested both 1h and O/N. Wash 3x.
- Stain with secondary antibodies + Phalloidin (abcam, iFluor680) + DAPI.
-Mount on a slide: we started with Prolong Diamond. Since we were having these issues we thought maybe we had a bad batch of Prolong, so we asked a bit around to test other mounting media, and we measured in parallel 2 different batches of Prolong Diamond, Fluoroshield and mowiol. The problem was the same everywhere.
- Let the mounting media harden O/N at RT. Seal with nailpolish, store at 4°C in a slides box.
We have double-checked the microscope settings with the people from the facility, we really don't think we are bleaching the samples in any unintended way since we do have signal the very first day, then the next day we almost don't, independently of whether we imaged the first day or not. Samples are always in the dark, we sre very careful not to have the lasers on unnecessarily.
Please any help would be greatly appreciated 😊