[Histonet] Re: Sections contamination after Lac Z staining

[Galina Deyneko]

Dear Colleagues
 I search your advices regarding Lac Z staining on frozen OCT embedded brain. After staining in Xgal staining solution overnight at 37C all sections covered by glassy small particles. The sections themselves are not contaminated, i checked them with H&E staining and also checked under microscope before placing in the beta gal staining solution. The blue Lac Z staining is good and intense.
Short method description:fix brain in freshly prepared Glutaraldehyde-Formaline fixative 2 hours, after Sucrose embed in OCT. Section thickness 7 microns. For staining I use Millipore kit. Of cause I warm the stain base solution at 37C before to dilute  melted beta Gal stock and the staining solution is clean and transparent, no precipitation. After staining fix sections in 10% formalin for 15 minutes and counterstain in Nuclear Fast red for 40 seconds.
I have try several modifications: no rinse the sections in the DI water, rinse the sections in DI water to get rid of OCT, after staining, and after counterstaining, nothing help. I am even  not able to catch the moment when appears this contamination. maybe filter the beta gal staining solution before placing slides? please share your suggestions, or detailed protocols.
 Thank you in advance 
Galina Deyneko
Novartis, Cambridge, MA
 
617-871-7613 w
 

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