Hugh wrote:
Sophia, Holes like you mentioned, are probably from the polymers or impurities in your embedding wax separating. Gayle Callus mentioned this several times (see histosearch), and recommended "Stirring" the embedding paraffin every so often (before every use). It could also be your tissue processing. Check to make sure it is properly processed. Embed without letting the wax solidify before doing so. You may have a bad batch of reagents? As for the stainer, Hazy nuclei, as long as you are sure your hemotoxylin is fine, are indicative of incomplete deparaffinzation, which could mean your OVEN or your reagents. It could also be your acid/base solutions. Note: Have you started a new lot # of wax? How about xylene (xylene-substiture) or alcohols? I have heard people saying X-brand paraffin was having problems with impurities that could cause these problems. I also heard it was solved last year. Also, your stainer has very little leeway in deparaffinzation. You might have to do things by hand until this problem is resolved. You know, deparaffinize to water using the longer method, then finish the H&E in the stainer. Does this help? Good luck (whoa, you have your work cut out for you), **************************************************************************** *********************** Hugh, Thank you for the kind words and your reply was right on the money. In fact, we stirred the paraffin daily before embedding, since the settling of polymers is not just occasional. Also, clean your embedding center frequently, before adding more paraffin. There is a clever little test for paraffin carryover into your rehydration alcohols when removing paraffin. 1) Use a glass beaker, add a few ml of used alcohol starting with last 95% just before 70% in deparaffinization setup. 2) Pipette a few mls of tap water into this aliquot of 95% and look for cloudiness. If a white cloud occurs, you have paraffin carryover all the way down your deparaffinization process. 3) If you don't see cloudy in this last 95%, test the 95% before this one, and keep going backwards towards the xylene or xylene substitute. If the next 95% is cloudy, there is paraffin carryover. Change out that alcohol station and all the ones before that including the xylene/xylene substitute. Also, you can do "rotation" where you move the second ( closest to water) 95% into first 95% spot, then replace the second 95% with fresh, replace all 100% and xylene/xyl sub as these latter are probably heavily contaminated with paraffin which is carrying over into your alcohols at some level. We used two or three xylene/xylene substitute changes (three preferred when doing IHC at 5 min/change) , two (or three) 100%, two 95% and one 70% alcohol changes before distilled water, 3 minutes per change, with hand staining. Change distilled water frequently, fresh daily and more changes when staining many slides as you don't need alcohol carry over into your hematoxylin. Careful monitoring of your solvents and distilled water should allow better hematoxylin staining. We also changed our acidic solution after hematoxylin and bluing solutions daily, these solutions are cheap plus always doing a 1 minute running water rinse after hematoxylin, acidic solution, and bluing. If you don't have running water rinsing, at least change your water rinses before each staining run. This all sounds very picky, but our H&E staining was very successful. Gayle Callis HTL/HT/MT(ASCP) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet