I recently did IHC staining of mouse brain tissues with four different primary antibodies, but am unsure as to how I am supposed to assess and troubleshoot these confocal images. The following images are labelled with the primary antibodies used to stain them. Also, all the images are presented together with the DAPI channel.
Hard to say without more information.
Brain tissue has a fair amount of autofluorescence, as do red blood cells. You may be seeing autofluorescence, but you'll only know for sure by looking at samples without antibody.
You could be seeing non specific staining of your secondary, but you'll only know by looking at samples without primary.
The punctate staining could be real, but it looks like antibody precipitate to me. I'll alway centrifuge my secondaries, vortex to mix, and centrifuge again. Then when I take some antibody, I only pull from the top of the solution to avoid any fluorescently tagged antibody that has precipitated.
Good luck!
Dear Mathangi.
I agree with Michael. You could test for autofluorescence and precipitating secondary by staining a control without primary, as Michael suggested.
Did you record a z-stack? Sometimes the last image near to the glassslide looks like in images 2 and 3. Maybe the other images from the z-stack look better.
Some tips on how to limit autofluorescence:
- If you stain paraffin sections, make sure that all paraffin is removed by incubating the slides for 4h in xylene, before rehydration steps for example. Paraffin is also a source of autofluorescence.
- Make sure that your slides don´t dry out during staining
- You could quench autofluorescence by incubating your slides in SudanBlack (0,3% in 70% EtOH) for 10 minutes before coverslipping.
May I ask what antibodies you use?
Hope this helps
Thank you both for you suggestions! Now I have a rough idea as to what I should troubleshoot. As for Mr Gasis, this is the order of antibodies I used for the images (from top to bottom): anti-iNOS (right-hemisphere), anti-CD11b, anti-iba1, anti-myelin CNPase, anti-iNOS (left hemisphere).
As for the recording a z-stack, I have not done it.
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