mouse tissue processing

[pisces22feb]

hi,

Can any one help me in deciphering the mistake in processing adult mouse skin and intestine for In Situ Hybridisation and histological staining. I am Fixing my tissue in 4% PFA overnight at 4 degree and then dehydrating in graded series of alcohol (25%,50%, 70%,80%,90% and 100%). Xylene is used as clearing for 3 minutes and 2min and then paraffin washes of 1 hour and overnight duration at 65degree, following embedding in paraffin. I am not getting good quality sections (5-7micro meters), tissue gets torn off from between the sections. Skin tissue section fall off from the slide during In Situ Hybridisation process.

Edited August 16, 2012 by pisces22feb

[alpha2cen]

Is the tissue size too large? Is xylene clearing time too short? Some alcohol might remain in the tissue.

I think that to read the experiment section in the paper more observantly will be good to solve this problem.

Edited August 18, 2012 by alpha2cen

[pisces22feb]

Is the tissue size too large? Is xylene clearing time too short? Some alcohol might remain in the tissue.

I think that to read the experiment section in the paper more observantly will be good to solve this problem.

 

Thanx for your reply. My sections are approximately 1cm and I do the xylene clearing untill the tissue is transparent and that too I give two washes of xylene.