You CAN NOT use water to dissolve the diastase. You have to use diastase buffer pH7 otherwise it will not work (as in your case). René J.
--- On Wed, 2/9/11, Erin Sarricks <esarri...@gmail.com> wrote: From: Erin Sarricks <esarri...@gmail.com> Subject: [Histonet] PAS with Diastase Digestion To: histonet@lists.utsouthwestern.edu Date: Wednesday, February 9, 2011, 7:38 AM Hi Histonet- I recently ran a PAS/D stain and had some issues with it. Both "with" and "without" slides came out looking the same so I'm guessing my digestion step didn't work! I used a Malt Diastase solution (0.5g to 500mL water) for my digestion. This is the procedure I used: 1. Deparaffinize and hydrate to water. 2. Place the sections labeled “with” in diastase solution preheated to 37˚C for 1 hour. Hold the sections labeled “without” in distilled water. 3. Wash in running water for 5 minutes 4. Place all section (with and without) in 0.5% periodic acid solution for 5 minutes 5. Wash in 3 changes of distilled water 6. Place in Schiff reagent for 15 minutes 7. Wash in lukewarm tap water for 5 minutes (immediately sections turn dark pink color). 8. Counterstain in Mayer’s Hematoxylin for 3 minutes. 9. Wash in tap water for 10 minutes 10. Dehydrate starting with 95% ETOH, clear, and coverslip. I am wondering if my solution possibly got too warm in the oven and hindered the enzyme activity, or is it possible I left it in too long? Any tips would be much appreciated! Oh, and I have about 300 slides to stain, so spitting on them is my last last last resort! Haha! Thanks in advance for all your help! Erin Sarricks, HT (ASCP) Histology Laboratory Technician USAMRICD Comparative Pathology Branch Office: Bldg E-3081 Room 178 E-mail: erin.p.sarri...@us.army.mil _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ____________________________________________________________________________________ Don't pick lemons. See all the new 2007 cars at Yahoo! Autos. http://autos.yahoo.com/new_cars.html _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet