Re: [Histonet] annoying crystals on sections
we are using a Sakura DRS2000 and we are 3 x 3 minutes in xylene and we have been keeping it fresh. The staining is good now but we still see the crystals. If it were paraffin we should see unstained spots on the slide I think. I have gone to an aqueous 1% HCl today after hematoxylin for regression and that seems to be cleaning them up on most of the slides. I cleaned out some of the plumbing and cleaned some calcium out of the pipes. We are using Harris hematoxylin that we purchase. We have a tried different counterstains but it seems to make no difference. We are using a Sakura tissue processor for overnight processing of cassettes. the embedding is going good and we get nice flat thin sections. We are fixing tissues with neutral phosphate buffered formalin but still see some formalin pigment. We are cleaning that up with picric acid in etoh. We find we still need that and the formalin pigment is brown to dark brown. These problem crystals are round irregular to rhomboidal some times sort of large and flat about the size of a cell and they are clear. I thought they were formalin pigment at first and fiddled with the Picric Acid, and tried Ammonia in Alcohol to get rid of formalin pigment and finally decided that it was not formalin pigment. I thought it might be something from Scott's tap water (Mg++) so i dropped that and tried bluing with NH4+ and it didnt help any. I tried blueing just with tap water. Nice result but still the crystals. The aqueous HCl seems to be working and is not harming the nuclei so I may have a sort of solution and am calling it calcium crystals in the water until I know better. I may look for some sort of filter to put in the water line. E Wayne Johnson DVM Enruikang Ag Tech MOA Feed Industry Centre China Agriculture University Beijing On 8/21/2012 7:51 PM, Debra Siena wrote: could it be paraffin? Debbie Siena HT(ASCP)QIHC Technical Manager | StatLab Medical Products 407 Interchange St. | McKinney, TX 75071 Direct: 972-436-1010 x229 | Fax: 972-436-1369 dsi...@statlab.com | www.statlab.com - Original Message - From: e...@pigsqq.org [mailto:e...@pigsqq.org] Sent: Tuesday, August 21, 2012 05:46 AM To: Debra Siena Subject: Re: [Histonet] annoying crystals on sections We just now ran the statlab version protocol from StatLab's website, using an alcoholic eosin. No doubt that gives a stronger brighter red stain. However we still see those crystals! We are suspecting a water problem. I have been dismantling some of the plumbing and getting some Ca crystals out of the pipes. We are manually coverslipping. ---Original Message--- From: Debra Sienadsi...@statlab.com To: 'e...@pigsqq.org'e...@pigsqq.org Subject: Re: [Histonet] annoying crystals on sections Sent: Aug 21 '12 07:46 Is your eosin alcoholic or aqueous? What is your staining protocol and what reagents are you using? This information would be most helpful. Also are the sections paraffin embedded and routinely processed in tissue processor? Debbie Siena HT(ASCP)QIHC Technical Manager | StatLab Medical Products 407 Interchange St. | McKinney, TX 75071 Direct: 972-436-1010 x229 | Fax: 972-436-1369 dsi...@statlab.com | www.statlab.com - Original Message - From: E. Wayne Johnson [mailto:e...@pigsqq.org] Sent: Monday, August 20, 2012 06:20 PM To: histonet@lists.utsouthwestern.eduhistonet@lists.utsouthwestern.edu Subject: [Histonet] annoying crystals on sections We are having problems with crystals precipitated on our slides which are HE stains on tissues from pigs. Tissues are fixed in buffered formalin. We had trouble months ago with formalin pigment and we had resolved that by using ammonia in EtOH or picric acid in EtOH. Sometimes we receive fixed samples from the field that are not buffered but presently all of our tissues are fixed in neutral phosphate buffered formalin. We moved the Sakura autostainer to a different location under a fume hood on a different floor of the building to get the solvent odor out of our work area. Immediately we began to see a tremendous degradation in slide quality due to what we initially thought was formalin pigment. We have changed all of the solutions and all of the stains. We find that if we use Milli-q water instead of tap water for rinsing (done by hand in that case) we dont see the crystals, but the eosin staining quality is not acceptable after rinsing in the acidic (ph ~5) Milli-Q water. Our tap water is neutral to slightly alkaline and is very hard with calcium. We do all sorts of tissues for diagnosis of pig diseases. Sometimes the slides are quite acceptable but sometimes particularly when looking at small intestine, the crystals are very annoying. The crystals occur randomly on the slide except that there is a tendency for them to be centered on nuclei particularly in intestinal epithelium. The crystals
RE: [Histonet] annoying crystals on sections
Hi Wayne, I had lots of problems with round irregular crystals, but have greatly improved my slides by limiting baking at 57 degrees to 1/2 hour. We use a paraffin with plastic in the mix, and I think the plastic globs up under some conditions, like no or not enough xylene to dissolve it out. Tom T -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of E. Wayne Johnson Sent: Tuesday, August 21, 2012 8:09 AM To: Debra Siena; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] annoying crystals on sections we are using a Sakura DRS2000 and we are 3 x 3 minutes in xylene and we have been keeping it fresh. The staining is good now but we still see the crystals. If it were paraffin we should see unstained spots on the slide I think. I have gone to an aqueous 1% HCl today after hematoxylin for regression and that seems to be cleaning them up on most of the slides. I cleaned out some of the plumbing and cleaned some calcium out of the pipes. We are using Harris hematoxylin that we purchase. We have a tried different counterstains but it seems to make no difference. We are using a Sakura tissue processor for overnight processing of cassettes. the embedding is going good and we get nice flat thin sections. We are fixing tissues with neutral phosphate buffered formalin but still see some formalin pigment. We are cleaning that up with picric acid in etoh. We find we still need that and the formalin pigment is brown to dark brown. These problem crystals are round irregular to rhomboidal some times sort of large and flat about the size of a cell and they are clear. I thought they were formalin pigment at first and fiddled with the Picric Acid, and tried Ammonia in Alcohol to get rid of formalin pigment and finally decided that it was not formalin pigment. I thought it might be something from Scott's tap water (Mg++) so i dropped that and tried bluing with NH4+ and it didnt help any. I tried blueing just with tap water. Nice result but still the crystals. The aqueous HCl seems to be working and is not harming the nuclei so I may have a sort of solution and am calling it calcium crystals in the water until I know better. I may look for some sort of filter to put in the water line. E Wayne Johnson DVM Enruikang Ag Tech MOA Feed Industry Centre China Agriculture University Beijing On 8/21/2012 7:51 PM, Debra Siena wrote: could it be paraffin? Debbie Siena HT(ASCP)QIHC Technical Manager | StatLab Medical Products 407 Interchange St. | McKinney, TX 75071 Direct: 972-436-1010 x229 | Fax: 972-436-1369 dsi...@statlab.com | www.statlab.com - Original Message - From: e...@pigsqq.org [mailto:e...@pigsqq.org] Sent: Tuesday, August 21, 2012 05:46 AM To: Debra Siena Subject: Re: [Histonet] annoying crystals on sections We just now ran the statlab version protocol from StatLab's website, using an alcoholic eosin. No doubt that gives a stronger brighter red stain. However we still see those crystals! We are suspecting a water problem. I have been dismantling some of the plumbing and getting some Ca crystals out of the pipes. We are manually coverslipping. ---Original Message--- From: Debra Sienadsi...@statlab.com To: 'e...@pigsqq.org'e...@pigsqq.org Subject: Re: [Histonet] annoying crystals on sections Sent: Aug 21 '12 07:46 Is your eosin alcoholic or aqueous? What is your staining protocol and what reagents are you using? This information would be most helpful. Also are the sections paraffin embedded and routinely processed in tissue processor? Debbie Siena HT(ASCP)QIHC Technical Manager | StatLab Medical Products 407 Interchange St. | McKinney, TX 75071 Direct: 972-436-1010 x229 | Fax: 972-436-1369 dsi...@statlab.com | www.statlab.com - Original Message - From: E. Wayne Johnson [mailto:e...@pigsqq.org] Sent: Monday, August 20, 2012 06:20 PM To: histonet@lists.utsouthwestern.eduhistonet@lists.utsouthwestern.edu Subject: [Histonet] annoying crystals on sections We are having problems with crystals precipitated on our slides which are HE stains on tissues from pigs. Tissues are fixed in buffered formalin. We had trouble months ago with formalin pigment and we had resolved that by using ammonia in EtOH or picric acid in EtOH. Sometimes we receive fixed samples from the field that are not buffered but presently all of our tissues are fixed in neutral phosphate buffered formalin. We moved the Sakura autostainer to a different location under a fume hood on a different floor of the building to get the solvent odor out of our work area. Immediately we began to see a tremendous degradation in slide quality due to what we initially thought was formalin pigment. We have changed all of the solutions and all of the stains. We find that if we
Re: [Histonet] annoying crystals on sections
Obviously, it's your water, if you don't see the crystals after changing your water. If the acidic pH of the Milli-q water is leaching out your eosin, then adjust the pH of the water to 7.0 before using it to rinse your slides. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
