I am trying FISH on mouse brain tissue, but I got cross reactivity between the miR signal and the antibody I used.
Brains are fixed with 4% PFA, cryopreserved and sectioned (12.5um thickness). I am using a DIG probe for a micro RNA, and the ISH for the selected probe is working. I want to localise the probe signal so I select a number of neuronal and astrocyte markers for that. However, when I did that I had cross-reactivity, despite using antibodies raised in different animals. I tried to use different blockings like 10% NGS or NDS and 1% BSA, still the same problem. Also, I changed the antibody, but with no help. When I stained the antibody alone, it worked!
I am using 50 degrees for Hibredisation and 60 degrees for washing in SSC.
Hi Hadil Alahdal,
Check this paper (http://www.nature.com/neuro/journal/v17/n11/full/nn.3813.html). They obtained beautiful results by combining FISH and IHC (they did some optimizations, and call their method TAI-FISH). Gook luck!
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