I have these squiggles that are appearing in my GAD67 IF stain and I'm not sure why. I've used this secondary antibody (Cy5 goat anti-mouse) with another primary antibody and never had this problem. In some areas, the squiggles aren't bad, but in others like the hippocampus, they're more prominent.
Any ideas?
EDIT: I don't know why the image is coming out blurry on here.
Hi Marcia,
Can you upload a clear image with arrows pointing to what you mean by "squiggles", please?
Sathya Srinivasan
Hi Marcia,
It's a bit hard to judge, given that the image appears blurry, but you should first determine if this background comes from the primary or the seconday antibody : check if you have the same staining when you omit the primary antibody.
I guess that you are working on mouse samples? If so, and if your primary antibody is made in mouse, there is a risk that it will be captured by Fc receptors, thus giving you non-specific staining. The secondary antibody (anti-mouse) can also cause background staining, since it can recognize endogenous mouse IgG, although this is usually not a problem in the hippocampus.
You said that you didn't have the same problem when using a different primary antibody, with the same secondary : did you do this experiment on slices that come from the same animal as the one you're having trouble with?
Sathya Srinivasan and Vincent Simon, hopefully this image looks better. Squiggles isn't the right word. It's more like bright spots that are thread like but fuzzy. The tissue I'm using here is rat whole coronal sections. I should add that this tissue is unfixed and frozen.
Vincent Simon , the tissue I'm working with is unfixed fresh froze rat tissue. My GAD67 primary antibody is from mouse and my secondary is from goat which is the same as the serum in my blocking solution.
I should have tried a secondary only condition but I didn't because I haven't had an issue with Cy5 like this before. I'll try that today.
The tissue I've been piloting this with has been from the same brain and this is the second time I've gotten background like this.
I'm beginning to think these could be blood vessels because the tissue wasn't fixed (not my choice). Someone elsewhere suggested it could be autofluorescence but I'd expect the background in general to be brighter and not have these brighter spots. If these are blood vessels, could they be autofluorescing?
Marcia Chavez, red blood cells are indeed autofluorescent. If the brain has not been perfused with PBS or saline before extraction and freezing, then this might be an issue.
In this case, you should check the endogenous autofluorescence of your tissue (just wash and mount some slices). Usually, red blood cells will "autofluoresce" in all channels, so take a picture in the green and red spectra as well, to see if you have the same type of signal. This, combined with the "secondary only" condition we discussed earlier will help you identify the problem.
Vincent Simon , thank you! I ended up checking for endogenous autofluorescence yesterday and there is none in the Cy5 channel. However, there is some in GFP. I'll be looking at my secondary only condition today to see what that looks like.
If it turns out that blood vessels are the problem, are there ways to remove or at least minimize their interference in a stain?
Edit: Secondary only conditions shows the "squiggles." I think these are blood vessels.
Marcia Chavez, I've read that Sudan Black B treatment can help with this kind of autofluorescence, although I have never tried it myself. Here is an article in which the authors compared different techniques to reduce autofluorescence and found that a 20-min incubation in 0.1% Sudan Black B dissolved in 70% Ethanol was the most effective. They did it in combination with FISH though, not IHC : https://core.ac.uk/download/pdf/37464562.pdf
There is also a commercially available compound named TrueVIEW, which acts as an autofluorescence quencher, and is supposed to be effective on red blood cell endogenous fluorescence : https://vectorlabs.com/products/blocking/trueview-autofluorescence-quenching-kit
I've tested TrueVIEW, and in my hands (brain slices from perfused mice), it decreases the background, but also greatly reduces the intensity of any specific staining. If you use Sudan Black B though, I've read somewhere that it can also increase the background in the red and far-red channels specifically, so beware of this.
Marcia Chavez, if you see the "squiggles" after incubating with the secondary antibody but not before, then the problem might not be related with red blood cell autofluorescence. Maybe your secondary antibody is captured by Fc receptors at the surface of immune cells within these blood vessels. It could also be possible that your anti-mouse antibody recognizes rat IgG, since they are quite similar.
You have different options then:
1) Use another primary antibody for GAD67, that is not made in mice.
2) If you want to keep your mouse primary antibody, then test an anti-mouse secondary antibody that has been cross-adsorbed against rat IgG and/or with only the F(ab')2 or Fab fragments. This might prevent any unwanted capture/binding of the secondary antibody. For instance this one, from Jackson Immuno, a goat F(ab')2 anti-mouse-AlexaFluor647, with minimal cross-reactivity with a lot of species, including rats: https://www.jacksonimmuno.com/catalog/products/715-606-151.
I may have missed this in the comments, but what type of species is the tissue? you used goat anti-mouse secondary in the past what is your secondary now? Your secondary antibody may be cross reacting with the tissue. are you using nail polish to keep the slides on? I have seen when nail polish leaked under the coverslip and creates this type of fluorescents. I recommend only painting the corners of the coverslip or at least look at the slide first before painting the coverslips with nail polish.