Hi all!
I have to immunolabel whole rat brains (and then clear them) for light sheet microscopy imaging and I will use, as primary antibody, anti-TH antibody AB1542 (Sheep) from Millipore. I can not find anything in the literature for this situation so, do you have any suggestion for the dilution that would be the best to use?
Thank you in advance
It appears there is only a recommendation by Millipore for Western Blot (the antibody may not work for immunofluorescence purposes). I would test the antibody on a brain slice at a dilution of 1:200 and 1:500 to start (the manufacturer recommends 1:1000 for western Blot--- usually IF applications require higher concentrations of antibody). Take a look at the sample under a confocal and see if it works. If not you may save yourself a lot of trouble as it certainly won't work for a cleared sample. Purchase another antibody and move from forward from there with similar optimization.
You can find the validated TH antibody for iDISCO protocol in this link, hope you find it useful. https://idisco.info/validated-antibodies/
I think you should standardize the antibody for your conditions and get the clue from the concentration given by the company (if at all given by the company). You may standardize using confocal or simple fluorescence microscopy as already mentioned previously. It may be a harder way but definitely the best. Good luck.
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