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, 201213 yr by 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.

Edited August 18, 201213 yr by 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.

 

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.