I've been trying to stain some mouse brain sections for GFAP, but got some disappointing results. I can see that the antibody works since there are some visible astrocytes, but not much else.
See attached for GFAP staining for someone's old sections from the lab (she has now left and I cannot get hold of her), and my staining. My staining appears sparse, and faint in comparison. See also attached my full protocol.
I have tried:
- Longer DAB incubation: the attached was incubated for 6 mins and the reaction stopped
- Changing reagents / antibody concentrations - all reagents are freshly prepared and pH balance
I don't have the time (or antibody stores) to keep changing things around and getting the same results! Does anyone recognise the appearance of my DAB staining and have any suggestions? Thank you very much.
After checking your immunohistochemical method and pictures, I think the immunohistochemical staining method is correct. Your picture 1 has astrocytes, and the number is really small. It may be that the background positive is too strong. If time permits, the materials can be taken from different parts. If time does not permit, if I am an editor, I think it is qualified, fig. 2 is actually a good sampling site, resulting in a rich number of astrocytes that can be found under a microscope. I also provided fig. 1 is GFAP staining applied by pathology department in the clinical manual, with positive staining of cytoplasm. pictures 2 and 3 are standard immunohistochemical manual staining. if it is still not ideal, immunohistochemical machine staining can be tried.
If you are confident in your protocol and your antibodies, then check the DAB. How old is it? How was it stored? Over time DAB looses some of its zip.
https://pmc.ncbi.nlm.nih.gov/articles/PMC11327071/pdf/fnagi-16-1397901.pdf
I think the problem is that you used mounted thick sections (20µm). I would recommand to use the free floating method. That garantees you an equal staining result because you incubate your sections in that way that the abntibodies coud pentrate the tissue from both sides. There is no need for microwave enhancement. In the paper which you find above there we discribed this method because we did a GFAP study. We have used PBS but this protocol works even with TBS. But befor you will start with your study check the protocol with different antibody dilutions an finally take the best after reconfirmation. If you have any questions please contact me again. good luck!
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