Dear all,
I plan to do western blot and Immuno-fluorescence against cholecystokinin and GAD65 using frozen mouse brains. Tissue for Immuno-fluorescence is fixed, cryo-protected and cut using cryostat. I have two questions:
1-Do you have suggestions for good antibodies that work in mouse tissue against CCK and GAD65 (not positive for GAD67)? I plan to do double immuno-fluorescence, so I should choose them in two different hosts.
2-For those who have used staining against cholecystokinin in mouse brain, is it that the antibody will stain only the axonal part or does it stain the soma as well?
Thanks
You must ask this question to immun stain sales firm. I don't have experience about this stains.
see our paper Schnorrbusch et al. (2013) Histochem. Cell Biol 140: 549-566 which might be useful.
Dear Abdelrahmanm , it is not easy to find suitable antibodies for mouse tissue. The GAD65 anitbodie which I used for long time is not available anymore. My favorite alternative is the GAD65 antibody which is selled by Synaptic Systems (www.sysy.com). Here you have the possibility of several different host animals. This will be helpful for the combination with CCK. www.antibodies-online.com offer different CCK-abs. You have to check which is the right one for your purpurses. Importend thing you have to care with when you are buying new antibodies: 1. reactivity (in youre case are the abs working on mouse tissue), 2. application (IHC, WB) and 3. the epitope of interest. Hope this will help you. Good luck!
You may use either a fluorescinated F(ab')2 as primary antibody or if you use a primary antibody, use fluorescinated F(ab')2 as secondary antibody, Fabs penetrate much better in tissue sections, either frozen or paraffin. PE or FITC as fluorescent dye is better.
yes use two different host,
otherwise both overlap,
try rat and goat
Hello Abdel,
I hope you got some answers to your original question. Just to add some from my side I would like to inform that there is a monoclonal antibody to GAD65 is available from Abcam company. The catalogue # is ab85866 and can be used for western blot, IP, flow cytometry etc. Even if the company has not tested this antibody for IH or IF I am sure you can use the same antibody for that purpose as well. Only thing is that you have to tag that antibody with a fluorophore that can be viewed through fluorescence microscope and in that case I would recommend any Alexa fluorophore from Molecular Probes. Abcam is a very trusted company. I have used their products time and again and got satisfactory results. Of course for double labeling you have to use the other antibody from a different species so they do not conflict each other. Good luck with your trials.
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