[Histonet] Help With In Situ


One of the Grad Students asked me to post her question. The Mouse Testes and Mouse Brain tissue for ISH Sox3 was kept at 4 degrees, 4% PFA fixed overnight, 30% sucrose cryoprotected until it sank, embedded in OCT, flash frozen. The ISH on the Mouse Brain was very good, but not the Mouse Testes. Two photos ISH Sox 3 Testes and ISH Sox 3 Brain are in the image gallery at histonet.org. 
 Thanks, Donna 

Here's Monica's question: 

I want to know if anyone else had experience with ISH on adult mouse testis cross-sections and if they've seen this background staining that specifically binds to elongated spermatid heads... And of course if anyone has been able to block against this. My sox3 probe is supposed to bind to the undifferentiated spermatogonia along the basement membrane of the seminiferous tubules. 
This staining appeared before the positive staining (approximately 3-4h 
after incubation) among the spermatid heads towards the lumen of the 
tubules. I have been using 4%PFA fixed testes sections and blocking with 
10% lamb serum. This staining has appeared with both an 820b probe and 440b 
Monica M Laronda 
Graduate Student, Jameson Lab 
Northwestern University 
Feinberg School of Medicine 
Chicago, IL 
Phone: 312-503-2036 

Donna J Emge, HT(ASCP)
Northwestern University 
Feinberg School of Medicine 
Chicago, IL 
Phone: 312-503-2036 
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