Sandy, After years of using Richard Allan's hematoxylin 2 with great success, if we didn't filter daily before use, we had stain precipitate on sections. Some of this comes from the hematoxylin continuing to oxidize in open air, bacteria and other "crud". Tim is absolutely correct ignoring manufacturers no filtering instructions. Being old school, we were taught to faithfully filter any hematoxylin, regardless of progressive or regressive types. If we topped off hematoxylin 2 or used new stock, the stain was filtered into a CLEAN staining container/dish. Keep an extra container around if possible. We used a medium fast filter paper, Whatman 54. I realize this takes time but junk on a slide is NOT good thing, especially after IHC staining and have a photo to show this - the result of being lazy and not filtering the hematoxylin on that particular day.
We used a distilled water rinse before hematoxylin2, but DI H2O will be contaminated with cellular debris and last hydrating alcohol carryover. Change DI water frequently if you have many runs in a day. We used 1 minute running tap water rinses after hematoxylin, clearant and bluing. If you are using running water rinses, take a look at the blue ppt in the post hematoxylin container as you don't want that sticking to sections. Non running water rinses should be changed after each H&E run in my opinion. Adequate clean water rinses are important to not have carry over of clearant into bluing reagents or bluing reagent into eosin in order to maintain correct pH for staining. Good luck Gayle M. Callis HTL/HT/MT(ASCP) -----Original Message----- From: Tim H via Histonet [mailto:[email protected]] Sent: Tuesday, September 22, 2015 11:25 AM To: [email protected] Subject: Re: [Histonet] Hematoxylin Precipitate You should be filtering your Hematoxylin on a daily basis regardless of what the manufactures says. We use to filter twice a day since we did a traditional overnight run and then again in the afternoon for specimens that had been microwave processed. So much tissue washes off in the solutions they should be changed or filtered fairly regularly to try and prevent cross contamination on the slides. You can also try increasing your rinse times and see if that doesn't help as well. Thanks, Tim > > Message: 1 > Date: Mon, 21 Sep 2015 15:14:39 -0500 > From: "Sandra Cheasty" <[email protected]> > To: "Histonet ([email protected])" > <[email protected]> > Subject: [Histonet] Hematoxylin Precipitate > Message-ID: <[email protected]> > Content-Type: text/plain; charset="us-ascii" > > Hello all, > > Has anyone using Richard Allen Hematoxylin-2 noticed an odd artifact on the slides after using the Hematoxylin for more than a few days on their stainer? We are seeing small spore or pollen-like blue dots here and there on the slides. It is not coming from the water bath or our water supply on the stainer. I used sterile gloves, opened a new case of slides, dipped them in DI water, then in the RA Hematoxylin 2 on the stainer, then in DI again, air-dried and coverslipped them, and the blue dots were there. The only way we got rid of the blue artifact was to use new RA Hematoxylin-2 every 2-3 days, which is a bit expensive. > > Thanks for your input, and if you can recommend a different, reasonably priced hematoxylin, that would be awesome. > > Cheers, > > Sandy > > > > Sandra J. Cheasty, HT (ASCP) > > Histology & Necropsy Supervisor > > UW-Madison, School of Veterinary Medicine _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
