I've been working on some FISH/IHC combo stains which have been working well until this last run where I got bright staining on the edge of the tissues but very minimal staining in the middle. The only difference this time was that I used PFA that I had made last week, aliquoted and frozen. I did some reading up on freezing PFA and I know there are mixed reviews. Does anyone have insight on what might have happened? I purchase the 16% PFA ampules so I don't mind making it up fresh every time; I was just trying to save the solution so I don't have to buy it all the time. I don't know what else it could have been. Thanks in advance for any tips!
Hi Adrienne,
What kind of tissue are you working on? (That migt be interesting to know to find a solution)
I also use frozen PFA (4%), but usually it works fine. How long do you fix with PFA? What proteins do you want to label?
I had a similar probem once, where the cell layer detached during the process of staining and re-attached folded (meaning 2 layers of cells stacked). It then appeared that I had brightly stained borders and no staining on the inside parts.
Best,
Anja
Hi Anja, thanks for your reply. I am working with fresh/frozen muscle biopsies. The protocol I have says to fix for 15 minutes at room temp. I ran 2 slides - one for the Staufen protein and the other for RBFOX1. With both slides I also ran FISH for 2´ O-methyl-CAG RNA probe. I did the FISH step before the IF. I've done these stains 3 or 4 times in the last few weeks and they've worked fine (I just started this job and had never done FISH or IF before). That's interesting that you said the tissue detached and re-attached because in-between each step, I like to look at the tissue and make sure it's still on the slide and I did notice that the tissue looked like maybe it was curling up off the slide - it was only slightly. I hate to do more experiments to test what happened because of how expensive everything is! But I would like to use the frozen PFA that I have aliquoted.
Hey Adrienne,
So maybe the curling up of the tissue might be a reason for te staining. Unfortunately I have no idea hw to solve that problem. Fixation duration seems fine, I do it te same way.
As I said above, I don't think that there's a problem with using frozen PFA, bt you might consider decreasing volume of aliquots to avid freeze-thaw-cycles.
Best
Anja
From what I can figure, you could be having fixation issues or permeabilisation issues... you've not stated if you permeabilise your cells? Also are you staining or labelling your cells? One could go on at length about the different aspects of sample prep for microscopy but I'll stick to PFA for now.
When one makes PFA, you're basically adding the powder to water and heating it up so that it depolymerises back to formaldehyde (this is what you fix your cells in). if you don't have stabilisers in it, it will eventually repolymerise back to PFA - this is the white precipitate you might see in the otherwise clear solution. Ideally, one should avoid this as you want to fix in formaldehyde. Did you notice any ppt forming when you reused the PFA?
What was the difference between the two pfa fixations? I'm guessing one was with freshly diluted PFA and the latter with the frozen aliquots? What percentage PFA are you using to fix in? Normally it's 3 or 4% PFA diluted in PBS. Also how did you freeze the PFA - We tend to snap freeze them down in LiN2, the reason being is you want to avoid the slow freezing of the water and PFA components of the PFA solution as they freeze at different speeds. Secondly, we tend to defrost our aliquots at 65-72C as it's known that PFA will ppt (polymerisation starts) at low temperatures so this avoids this happening, so basically you know that you have exactly the % of formaldehyde you have made up.
Also do you quench your fixation after incubation? We use Ammonium chloride solution for this.
Finally, just to point out something one needs to consider when buying in premade PFA solutions, it normally comes with a stabiliser added (normally methanol) - while this may not be an issue for some (light or confocal microscopy), it is not recommended for EM work as organic solvents will permeabilise/destroy the cell membrane which one wants intact for EM imaging.
Below is a typical protocol I use:
Fix cells with 3% (w/v) PFA in PBS. Made up fresh from 24% PFA stock solution which is stored at –20°C in aliquots and thawed @ 65°C. Incubate 20-30min at RT, aspirate fix and add quench solution (50mM NH4Cl in PBS). If I want to permeabilise my cells, I will add 0.2% (w/v) saponin in PBS to the quench solution. I then add my antibodies or stains direct to PBS with 0.2% saponin and either 0.2% gelatin or 5-10% serum (but not the same host as your antibody).
Sorry it's a bit long winded, but hope it helps!
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