I need to get good images of astrocytes in the hypothalamus, but the antibodies I have been using are not labeled very well, while the DAPI and my protein of interest appear perfectly. What dilutions do you usually use from each one?
Heya
I routinely use the following two antibodies:
- mouse anti-GFAP (clone GA5, Millipore MAB3402 - http://www.emdmillipore.com/GB/en/product/Anti-Glial-Fibrillary-Acidic-Protein-Antibody%2C-clone-GA5,MM_NF-MAB3402 or Novus NBP2-29415 - http://www.novusbio.com/GFAP-Antibody-GA5_NBP2-29415.html)
- rabbit anti-GFAP (Novus NB300-141 - http://www.novusbio.com/GFAP-Antibody_NB300-141.html)
They work great in culture as well as in rat and human sections. Start using them at 1:500, that should work.
In terms of secondaries, I usually use Biotium donkey anti-mouse CF568-tagged (https://biotium.com/product/donkey-anti-mouse-igg-hl-highly-cross-adsorbed-7-2/). I also buy secondaries from Thermo Fisher or Jackson ImmunoResearch, mostly Alexa-tagged. It all depends on your antibody combinations and laser lines.
Hope this helps. Good luck!
Hi;
The mouse anti-GFAP antibody from Merck Millipore (1/400) and then a fluorescent Alexa fluor 488- or 568-conjugated secondary antibody from Life technologies (1/200).
S.
Hi,
we are using the mouse anti-GFAP (Sigma-Aldrich, G3893; 1:400) or the rabbit anti-GFAP (DAKO, Z0334; 1:500 - 1:1000) antibody in combination with Cy3 or Cy5-conjugated antibodies from Jackson Immuno Research. With both antibodies, we can detect GFAP in brains from mouse or rat without any pre-treatments.
Good Luck!
S.
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