I am staining mouse brain tissue sections using an anti-GFAP primary antibody (for astrocytes). The results are coming out pretty weird. Some sections have decent staining, and some others have horrible/very weak staining. I'm using the same protocol and reagents as I usually do (and have had successful staining in the past), so I won't go into those details here.
Instead, this was my first time transcardially perfusing the animals with paraformaldehyde. I suspect some of the perfusions did not go well: a couple of the bodies did not get very stiff. After brain extractions, I put them in a PFA/sucrose solution overnight. Looking at my stained sections under the microscope, the clearance of blood didn't seem to be a huge issue. There is a little autofluorescence going on (due to the blood, I suspect), but overall, there is no blood in the sections.
So, could it be that the PFA didn't penetrate my tissue well? Would this cause extremely weak signal in my sections (even though the tissue did sit in PFA overnight)?
The pictures are examples.
1) The staining came out as expected on this one.
2) Verrrry weak signal, but you can see the GFAP.
3) An example of autofluorescence from the blood that was left. I see absolutely no GFAP/astrocytes.
Hello Laura,
generally we can say your immuno has worked. You see a positiv signal. The reason why your result looks different from those of your resent experiments is that you have changed the conditions. And now you have to ajust your protocol. That means you have to play with antibody concentrations as well as with blocking and dilution media.
To get an information about your fixation quality I recommand to do a Nissl's Cresyl violet stain. This will help you to find out wether the morphology of you tissue looks good or not.
I would recommand to do the cryprotection in 30% sucrose only after 24 h post fixation in PFA.
Another thing is the backroud staining . It is known that aldehyd fixation increases the autofluorescence especially in the green Channel (488 nm).
In that case it could be helpful to use another fluorophore like Cy3 (546 nm) or Cy5 (647 nm). Good luck!