I currently trying to perform a FISH protocol in some mouse samples, 4% PFA fixed and embedded in paraffin.
The probe and protocol I currently using are working but the results are not optimal since I get some variability in the results (mainly the background, that sometimes is very intense but if I do more washes or longer ones I sometimes lose my probe signal at all) even when using the exact same protocol and sections of the same sample.
I was wondering if anyone had preform FISH under similar conditions and what probe did you use.
Many thanks,
Joana
Are you looking for the chromosome itself or the presence of the Y? You could try Kdm5d, which is located on the Y. Here is a probe for the mRNA.
http://mouse.brain-map.org/experiment/show/70303606
https://genome.ucsc.edu/cgi-bin/hgTracks?db=mm10&position=chrY%3A897788-943811&hgsid=439247517_7aDPzaHNJffMDU6xRWhLwim0x9KS
Hi Liz Unger,
Many thanks for your help.
So what I need is to identify the male cells ina mosaic tissue of female and male mouse cells. Now I'm using a probe to detect the chromossome itself but this probe already come conjugated with a flurochrome and sometimes the backgound is not as clear as I would like...
Liz's suggestion of KDM5D is a good Y-specific gene. There are other options: the mouse Y long arm is composed of highly repetitive sequence; there are ~150-200 copies of a ~500kb tract called the 'huge repeat array'. This tract is itself internally repetitive, and includes the genes Orly, Ssty1 and Ssty2.
Orly: http://www.ncbi.nlm.nih.gov/gene/75204
Ssty1: http://www.ncbi.nlm.nih.gov/gene/20611
Ssty2: http://www.ncbi.nlm.nih.gov/gene/70009
These would be good targets for probe design, since you will get a very strong signal from even a small probe.
Hi
Does anyone know if there is an antibody to recognize Y chromosome specific proteins expressed in somatic cells from males?
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