RE: [Histonet] air drying special stain slides rather than dehydrate and clear
I suppose my age will show here but I was always taught to NEVER let slides dry out unless the procedure indicated such. Is there no drying artifact when you let these slide dry before coverslipping? Hazel Horn Supervisor of Histology/Autopsy/Transcription Anatomic Pathology Arkansas Children's Hospital 1 Children's Way | Slot 820| Little Rock, AR 72202 501.364.4240 direct | 501.364.1302 office | 501.364.1241 fax hor...@archildrens.org archildrens.org 100 YEARS YOUNG! JOIN THE PARTY AT ach100.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amos Brooks Sent: Tuesday, September 11, 2012 7:31 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] air drying special stain slides rather than dehydrate and clear Hi, My choice to air dry rather than dehydrate in ETOH xylene is based on the stain rather than the spooky xylene hazard boogyman. Yes, not using xylene if it is not really needed is not a bad idea, but the main reason I air dry some stains is the alcohols remove some of the stains. Ever have a beautiful Luxol Fast Blue bleach out on you? The most exasperating thing in the world! Generally stains that end in water can easily be air dried. Something alcoholic like eosin or Movat's Pentachrome ending in alcoholic saffron might as well be finished traditionally. I air dry any stain that is counterstained in Nuclear Fast Red, Light Green, Methyl Green. I have air dried IHCs with no ill effects too. Don't try it with fluorescents though, that would be bad ... and pointless. I don't put them in an oven. I set them at the front of the fume hood and go do something else for a few minutes. If I want to rush it I close the sash to increase the flow rate for a bit. (Of course it is opened back up right after so the draft works properly.) Amos On Tue, Sep 11, 2012 at 11:09 AM, histonet-requ...@lists.utsouthwestern.edu wrote: Message: 16 Date: Tue, 11 Sep 2012 10:32:08 -0400 From: Diana McCaig dmcc...@ckha.on.ca Subject: [Histonet] air drying special stain slides rather than dehydrate and clear To: histonet@lists.utsouthwestern.edu Message-ID: dcfd9e6a390e294aaf3a2561cd32e5c417a90...@ckhamail1.ckha.on.ca Content-Type: text/plain; charset=us-ascii I was hoping to get information on why special stains are dehydrated, cleared and mounted vs allowing them to be blotted dry, air dried then coverslip. Every procedure I have ever encountered always indicates to dehydrate and clear but I have heard where some labs are blotting the slides , allowing to air dry (probably not set standard time) and dipped in xylene prior to cover slipping. Reason given is that the counterstain gets washed out. Wouldn't adjusting the times be a better resolution. I understand residual water could be present and cause long term issues on storage but wanted some other opinions on this process. Diana ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ** The information contained in this message may be privileged and confidential and protected from disclosure. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please notify us immediately by replying to the message and deleting it from your computer. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RE: air drying special stain slides rather than
Dishwashing machines are not at all common in China even in Beijing so we could not find dishwasher detergent nearby. But we took a bus and subway ride toward the city center to Zhongguancun (the computer district) and found a Carrefour's (Jia Le Fu 家乐福) that had one brand of powdered detergent Finish (Reckett Benckiser). Finish was formerly called Electrasol. Actually I was a bit afraid of Finish. If I had known it was the same thing as the familiar Electrasol, I would not have had any concerns. Anyway, we took the Finish powder back to the lab where we had a covered water bath waiting at 90C. I mixed 40 grams in with 2L of tap water and we followed the protocol Rene' sent with some test tissues from some pigs we examined a few days ago (lung, liver, heart, spleen, kidney, gut). We heated the detergent solution on an induction stove and poured in into some square glass jars in the water bath. The procedure took the paraffin right off. We did an HE and dried the slide in the 60C oven after a water wash to clean up after Eosin. Ver-r-ry nice result. Jane tried the technique then by herself with 3 more slides including one slide with some honking big pieces of pig cerebellum. The sections all stayed put on the slides. Sometimes we can lose most of the cerebellum in processing, so we think it is a good demonstration that section loss is not going to be much of a problem. The stain was a Harris hematoxylin regressed with 1% HCl. We blued some with tap water and some with Scott's. I sort of prefer the plain tap water bluing. Our Eosin is an Eosin/Biebrich Scarlet (Acid Red 66)/Orange G that we make up. We usually use a treatment in alcoholic Picric Acid to get rid of formalin pigment but we omitted that step today and the slides turned out well. Indeed these pigs were a field necropsy and the formalin was simple 10% unbuffered formalin in plain local farm tap water, but there were only some traces of formalin pigment. We are wondering if perhaps the detergent is taking out some of the formalin pigment for us. We got a few white paraffin spots on one slide but even that would not be an issue in reading or photographing the slide. Jane thinks she can tweak the procedure to eliminate the paraffin spots. Jane's opinion on the procedure? She will be bottling up all of the xylenes and alcohols and storing them away first thing tomorrow morning. This fixes a big problem for us because the histolab is on the first floor along with some offices. We do our work under a fume hood and we are careful, but we had an incident where students left containers of xylene uncapped outside the hood overnight in hot weather vaporizing a large amount of xylene into the hallway. Not cool. We moved the tissue processor and autostainer to a remoter spot on the 4th floor but the water quality issues there made our autostainer a problem. We can now bring our autostainer back and set it up for special and routine stains. The procedure with detergent from beginning to end is significantly shorter than the xylene alcohols stain alcohols xylene procedure and we will dramatically reduce our consumption and waste output of xylene and our consumption of ethanol. Very cool. Thanks EW Johnson Enruikang Ag Tech Beijing On 9/12/2012 3:13 AM, Rene J Buesa wrote: EWayne: All mounting media contains a so called solvent. Permount contains toluene (not as nasty as xylene) and will penetrate the section provided it is absolutely dehydrated (in an oven in this case). You just have to finish the HC (or IHC) protocol and pop-in the slides in the oven. Balsam of Canada (the resin) is dissolved in xylene (always) so the penetration is also assured. Under separate cover I am sending you my articles. Try this method, you will love it! René J. *From:* E. Wayne Johnson e...@pigsqq.org *To:* Rene J Buesa rjbu...@yahoo.com *Cc:* Mayer,Toysha N tnma...@mdanderson.org; 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu *Sent:* Tuesday, September 11, 2012 1:14 PM *Subject:* Re: [Histonet] RE: air drying special stain slides rather than I am convinced to give it a try because I also have trouble will the loss of some stains in dehydration. I was concerned that the slides would not clear well after oven dehydration. I will see how it works for me. I can see clearly how going from counterstain to oven will save much hassle with xylene and alcohols as well as not washing out some special stains. I have tried some of the isopropyl alcohol and acetone dehydration called for in some of the stain procedures and it would be great if the slides could just be popped into the oven. What mounting medium are you using? Does it matter? I am a bit worried about penetration of the mountant into the tissue section if there is no xylene in the tissue. Will neutral balsam still work ok? Rene: if you have a link to the paper you talked about on eliminating xylene, I am interested.
[Histonet] Re: dissecting pins
Kimberly D. Kolman, HT, (ASCP) at the VA center in Leavenworth KS asks: Does anyone know where I can find some large-head pins to use when pinning out large tissue specimens for fixation? We already use the large T-pins and the regular bulletin board push-pins are too small. We need something with a larger, easier to grip plastic head, and long (at least ½ inch) pin shaft. - Thanks for your help (and hope it might be one of our 'contract vendors', or I won't be able to use them anyway... L) Go to any of the big stores that sell sewing supplies - they have innumerable varieties of pins - and pick out what you want. This is one of a number of items used in surgical grossing that you have to buy cheaply on the open market or scrounge, like hacksaws for cutting bone, plastic rulers, metal skewers - I have a whole kit bag of such items that I carry to my various jobs. Bob Richmond Samurai Pathologist Maryville TN ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RE: air drying special stain slides rather than
EWayne (et al): So, there you have it! He (or she) who still uses xylene in the histology lab is just because he (or she) has decided to do so! At this moment what you describe is standard procedure for several private labs in the US and the US Virgin Islands, Canada, Russia and Spain. Besides dewaxing with dishwasher soap and air drying before cover-slipping, you can also eliminate xylene from tissue processing by just following the instructions outlinedin the articles I sent. you. Try to contact as many colleagues as you can and spread the word: xylene is out of our lives, as long as we want to. Thank you for the information René J. From: E. Wayne Johnson e...@pigsqq.org To: Rene J Buesa rjbu...@yahoo.com Cc: Mayer,Toysha N tnma...@mdanderson.org; 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu Sent: Wednesday, September 12, 2012 9:49 AM Subject: Re: [Histonet] RE: air drying special stain slides rather than Dishwashing machines are not at all common in China even in Beijing so we could not find dishwasher detergent nearby. But we took a bus and subway ride toward the city center to Zhongguancun (the computer district) and found a Carrefour's (Jia Le Fu 家乐福) that had one brand of powdered detergent Finish (Reckett Benckiser). Finish was formerly called Electrasol. Actually I was a bit afraid of Finish. If I had known it was the same thing as the familiar Electrasol, I would not have had any concerns. Anyway, we took the Finish powder back to the lab where we had a covered water bath waiting at 90C. I mixed 40 grams in with 2L of tap water and we followed the protocol Rene' sent with some test tissues from some pigs we examined a few days ago (lung, liver, heart, spleen, kidney, gut). We heated the detergent solution on an induction stove and poured in into some square glass jars in the water bath. The procedure took the paraffin right off. We did an HE and dried the slide in the 60C oven after a water wash to clean up after Eosin. Ver-r-ry nice result. Jane tried the technique then by herself with 3 more slides including one slide with some honking big pieces of pig cerebellum. The sections all stayed put on the slides. Sometimes we can lose most of the cerebellum in processing, so we think it is a good demonstration that section loss is not going to be much of a problem. The stain was a Harris hematoxylin regressed with 1% HCl. We blued some with tap water and some with Scott's. I sort of prefer the plain tap water bluing. Our Eosin is an Eosin/Biebrich Scarlet (Acid Red 66)/Orange G that we make up. We usually use a treatment in alcoholic Picric Acid to get rid of formalin pigment but we omitted that step today and the slides turned out well. Indeed these pigs were a field necropsy and the formalin was simple 10% unbuffered formalin in plain local farm tap water, but there were only some traces of formalin pigment. We are wondering if perhaps the detergent is taking out some of the formalin pigment for us. We got a few white paraffin spots on one slide but even that would not be an issue in reading or photographing the slide. Jane thinks she can tweak the procedure to eliminate the paraffin spots. Jane's opinion on the procedure? She will be bottling up all of the xylenes and alcohols and storing them away first thing tomorrow morning. This fixes a big problem for us because the histolab is on the first floor along with some offices. We do our work under a fume hood and we are careful, but we had an incident where students left containers of xylene uncapped outside the hood overnight in hot weather vaporizing a large amount of xylene into the hallway. Not cool. We moved the tissue processor and autostainer to a remoter spot on the 4th floor but the water quality issues there made our autostainer a problem. We can now bring our autostainer back and set it up for special and routine stains. The procedure with detergent from beginning to end is significantly shorter than the xylene alcohols stain alcohols xylene procedure and we will dramatically reduce our consumption and waste output of xylene and our consumption of ethanol. Very cool. Thanks EW Johnson Enruikang Ag Tech Beijing On 9/12/2012 3:13 AM, Rene J Buesa wrote: EWayne: All mounting media contains a so called solvent. Permount contains toluene (not as nasty as xylene) and will penetrate the section provided it is absolutely dehydrated (in an oven in this case). You just have to finish the HC (or IHC) protocol and pop-in the slides in the oven. Balsam of Canada (the resin) is dissolved in xylene (always) so the penetration is also assured. Under separate cover I am sending you my articles. Try this method, you will love it! René J. From: E. Wayne Johnson mailto:e...@pigsqq.org To: Rene J Buesa mailto:rjbu...@yahoo.com Cc: Mayer,Toysha N mailto:tnma...@mdanderson.org; mailto:'histonet@lists.utsouthwestern.edu'
Re: [Histonet] air drying special stain slides rather than dehydrate and clear
Hazel: The so called drying artifact that appears as a darker granulation, like sand grains, is caused NOT by drying the sections, but by drying them incompletely. IF there is any remnant of water within or between the cells, that water will refract the light using to observe the sections and will appear as dark granules. It is the same effect as when you observe rain falling in the horizon: even when the water is transparent, you will see the falling rain as a darker film, almost black, also because of light refraction. If you completely eliminate the water, that artifact will not take place. Under separate cover I am sending something about this I published. By the way, I was taught the same when I started to study histology during my pre-medical studies in 1952 (so do not talk about age, I am for sure older than you are!). Keep your brain young, it does not matter if your body ages, your brain has to remain young! René J. From: Horn, Hazel V hor...@archildrens.org To: 'Amos Brooks' amosbro...@gmail.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Wednesday, September 12, 2012 8:57 AM Subject: RE: [Histonet] air drying special stain slides rather than dehydrate and clear I suppose my age will show here but I was always taught to NEVER let slides dry out unless the procedure indicated such. Is there no drying artifact when you let these slide dry before coverslipping? Hazel Horn Supervisor of Histology/Autopsy/Transcription Anatomic Pathology Arkansas Children's Hospital 1 Children's Way | Slot 820| Little Rock, AR 72202 501.364.4240 direct | 501.364.1302 office | 501.364.1241 fax hor...@archildrens.org archildrens.org 100 YEARS YOUNG! JOIN THE PARTY AT ach100.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amos Brooks Sent: Tuesday, September 11, 2012 7:31 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] air drying special stain slides rather than dehydrate and clear Hi, My choice to air dry rather than dehydrate in ETOH xylene is based on the stain rather than the spooky xylene hazard boogyman. Yes, not using xylene if it is not really needed is not a bad idea, but the main reason I air dry some stains is the alcohols remove some of the stains. Ever have a beautiful Luxol Fast Blue bleach out on you? The most exasperating thing in the world! Generally stains that end in water can easily be air dried. Something alcoholic like eosin or Movat's Pentachrome ending in alcoholic saffron might as well be finished traditionally. I air dry any stain that is counterstained in Nuclear Fast Red, Light Green, Methyl Green. I have air dried IHCs with no ill effects too. Don't try it with fluorescents though, that would be bad ... and pointless. I don't put them in an oven. I set them at the front of the fume hood and go do something else for a few minutes. If I want to rush it I close the sash to increase the flow rate for a bit. (Of course it is opened back up right after so the draft works properly.) Amos On Tue, Sep 11, 2012 at 11:09 AM, histonet-requ...@lists.utsouthwestern.edu wrote: Message: 16 Date: Tue, 11 Sep 2012 10:32:08 -0400 From: Diana McCaig dmcc...@ckha.on.ca Subject: [Histonet] air drying special stain slides rather than dehydrate and clear To: histonet@lists.utsouthwestern.edu Message-ID: dcfd9e6a390e294aaf3a2561cd32e5c417a90...@ckhamail1.ckha.on.ca Content-Type: text/plain; charset=us-ascii I was hoping to get information on why special stains are dehydrated, cleared and mounted vs allowing them to be blotted dry, air dried then coverslip. Every procedure I have ever encountered always indicates to dehydrate and clear but I have heard where some labs are blotting the slides , allowing to air dry (probably not set standard time) and dipped in xylene prior to cover slipping. Reason given is that the counterstain gets washed out. Wouldn't adjusting the times be a better resolution. I understand residual water could be present and cause long term issues on storage but wanted some other opinions on this process. Diana ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RE: air drying special stain slides rather than
EWayne (et al): So, there you have it! He (or she) who still uses xylene in the histology lab is just because he (or she) has decided to do so! At this moment what you describe is standard procedure for several private labs in the US and the US Virgin Islands, Canada, Russia and Spain. Besides dewaxing with dishwasher soap and air drying before cover-slipping, you can also eliminate xylene from tissue processing by just following the instructions outlinedin the articles I sent. you. Try to contact as many colleagues as you can and spread the word: xylene is out of our lives, as long as we want to. Thank you for the information René J. From: E. Wayne Johnson e...@pigsqq.org To: Rene J Buesa rjbu...@yahoo.com Cc: Mayer,Toysha N tnma...@mdanderson.org; 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu Sent: Wednesday, September 12, 2012 9:49 AM Subject: Re: [Histonet] RE: air drying special stain slides rather than Dishwashing machines are not at all common in China even in Beijing so we could not find dishwasher detergent nearby. But we took a bus and subway ride toward the city center to Zhongguancun (the computer district) and found a Carrefour's (Jia Le Fu 家乐福) that had one brand of powdered detergent Finish (Reckett Benckiser). Finish was formerly called Electrasol. Actually I was a bit afraid of Finish. If I had known it was the same thing as the familiar Electrasol, I would not have had any concerns. Anyway, we took the Finish powder back to the lab where we had a covered water bath waiting at 90C. I mixed 40 grams in with 2L of tap water and we followed the protocol Rene' sent with some test tissues from some pigs we examined a few days ago (lung, liver, heart, spleen, kidney, gut). We heated the detergent solution on an induction stove and poured in into some square glass jars in the water bath. The procedure took the paraffin right off. We did an HE and dried the slide in the 60C oven after a water wash to clean up after Eosin. Ver-r-ry nice result. Jane tried the technique then by herself with 3 more slides including one slide with some honking big pieces of pig cerebellum. The sections all stayed put on the slides. Sometimes we can lose most of the cerebellum in processing, so we think it is a good demonstration that section loss is not going to be much of a problem. The stain was a Harris hematoxylin regressed with 1% HCl. We blued some with tap water and some with Scott's. I sort of prefer the plain tap water bluing. Our Eosin is an Eosin/Biebrich Scarlet (Acid Red 66)/Orange G that we make up. We usually use a treatment in alcoholic Picric Acid to get rid of formalin pigment but we omitted that step today and the slides turned out well. Indeed these pigs were a field necropsy and the formalin was simple 10% unbuffered formalin in plain local farm tap water, but there were only some traces of formalin pigment. We are wondering if perhaps the detergent is taking out some of the formalin pigment for us. We got a few white paraffin spots on one slide but even that would not be an issue in reading or photographing the slide. Jane thinks she can tweak the procedure to eliminate the paraffin spots. Jane's opinion on the procedure? She will be bottling up all of the xylenes and alcohols and storing them away first thing tomorrow morning. This fixes a big problem for us because the histolab is on the first floor along with some offices. We do our work under a fume hood and we are careful, but we had an incident where students left containers of xylene uncapped outside the hood overnight in hot weather vaporizing a large amount of xylene into the hallway. Not cool. We moved the tissue processor and autostainer to a remoter spot on the 4th floor but the water quality issues there made our autostainer a problem. We can now bring our autostainer back and set it up for special and routine stains. The procedure with detergent from beginning to end is significantly shorter than the xylene alcohols stain alcohols xylene procedure and we will dramatically reduce our consumption and waste output of xylene and our consumption of ethanol. Very cool. Thanks EW Johnson Enruikang Ag Tech Beijing On 9/12/2012 3:13 AM, Rene J Buesa wrote: EWayne: All mounting media contains a so called solvent. Permount contains toluene (not as nasty as xylene) and will penetrate the section provided it is absolutely dehydrated (in an oven in this case). You just have to finish the HC (or IHC) protocol and pop-in the slides in the oven. Balsam of Canada (the resin) is dissolved in xylene (always) so the penetration is also assured. Under separate cover I am sending you my articles. Try this method, you will love it! René J. From: E. Wayne Johnson mailto:e...@pigsqq.org To: Rene J Buesa mailto:rjbu...@yahoo.com Cc: Mayer,Toysha N mailto:tnma...@mdanderson.org; mailto:'histonet@lists.utsouthwestern.edu'
[Histonet] Cryosectioning question
Hi All, I have a question about cryopresevation/ sectioning. I have a study (mouse kidneys sample)that I need to re-section the cryoblocks but the intern that sectioned them first did not reseal them with OCT so they have now seperated from the OCT (dessicated). I can't get sections because of the tissue and OCT separate. Can I re-freeze them? If so how do I avoid thawing the sample. Has anyone run into this, if so what did you do. Also is there a good method for sectioning out there I want to improve my speed and quality of cryosections so I hope to adopt some new tricks. Do people like using a camel hair brush or the anti-roll plate, I use a brush.. Is there any good refference material I should look into... Any help is appreciated. Thanks, Jamie ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Re: dissecting pins
Thanks everyone for all the replies. I've been perusing the office supply websites but had not thought of sewing supplies - I'll check that out! It will be a challenge to find the items my docs here will adjust to, also to get Supply to order hypodermic needles for this use; I am fighting with them now to get them to order non-sterile 4x4 gauze. Some nice inspection person has insisted to them that all gauze must be sterile.so now I can spend my time ripping open individual sterile 4x4 packs to have ready for coverslipping, cleaning the embedding center, etc nothing better to do with my time, I guess...! -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Bob Richmond Sent: Wednesday, September 12, 2012 8:51 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Re: dissecting pins Kimberly D. Kolman, HT, (ASCP) at the VA center in Leavenworth KS asks: Does anyone know where I can find some large-head pins to use when pinning out large tissue specimens for fixation? We already use the large T-pins and the regular bulletin board push-pins are too small. We need something with a larger, easier to grip plastic head, and long (at least ½ inch) pin shaft. - Thanks for your help (and hope it might be one of our 'contract vendors', or I won't be able to use them anyway... L) Go to any of the big stores that sell sewing supplies - they have innumerable varieties of pins - and pick out what you want. This is one of a number of items used in surgical grossing that you have to buy cheaply on the open market or scrounge, like hacksaws for cutting bone, plastic rulers, metal skewers - I have a whole kit bag of such items that I carry to my various jobs. Bob Richmond Samurai Pathologist Maryville TN ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] air drying special stain slides rather than dehydrate and clear
Hazel, The only time I have seen any problem was when a stain was not fully washed off the slide. Once you put the slide in xylene and coverslip it the slide is just like any other. Amos On Sep 12, 2012 8:57 AM, Horn, Hazel V hor...@archildrens.org wrote: I suppose my age will show here but I was always taught to NEVER let slides dry out unless the procedure indicated such. Is there no drying artifact when you let these slide dry before coverslipping? Hazel Horn Supervisor of Histology/Autopsy/Transcription Anatomic Pathology Arkansas Children's Hospital 1 Children's Way | Slot 820| Little Rock, AR 72202 501.364.4240 direct | 501.364.1302 office | 501.364.1241 fax hor...@archildrens.org archildrens.org 100 YEARS YOUNG! JOIN THE PARTY AT ach100.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto: histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amos Brooks Sent: Tuesday, September 11, 2012 7:31 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] air drying special stain slides rather than dehydrate and clear Hi, My choice to air dry rather than dehydrate in ETOH xylene is based on the stain rather than the spooky xylene hazard boogyman. Yes, not using xylene if it is not really needed is not a bad idea, but the main reason I air dry some stains is the alcohols remove some of the stains. Ever have a beautiful Luxol Fast Blue bleach out on you? The most exasperating thing in the world! Generally stains that end in water can easily be air dried. Something alcoholic like eosin or Movat's Pentachrome ending in alcoholic saffron might as well be finished traditionally. I air dry any stain that is counterstained in Nuclear Fast Red, Light Green, Methyl Green. I have air dried IHCs with no ill effects too. Don't try it with fluorescents though, that would be bad ... and pointless. I don't put them in an oven. I set them at the front of the fume hood and go do something else for a few minutes. If I want to rush it I close the sash to increase the flow rate for a bit. (Of course it is opened back up right after so the draft works properly.) Amos On Tue, Sep 11, 2012 at 11:09 AM, histonet-requ...@lists.utsouthwestern.edu wrote: Message: 16 Date: Tue, 11 Sep 2012 10:32:08 -0400 From: Diana McCaig dmcc...@ckha.on.ca Subject: [Histonet] air drying special stain slides rather than dehydrate and clear To: histonet@lists.utsouthwestern.edu Message-ID: dcfd9e6a390e294aaf3a2561cd32e5c417a90...@ckhamail1.ckha.on.ca Content-Type: text/plain; charset=us-ascii I was hoping to get information on why special stains are dehydrated, cleared and mounted vs allowing them to be blotted dry, air dried then coverslip. Every procedure I have ever encountered always indicates to dehydrate and clear but I have heard where some labs are blotting the slides , allowing to air dry (probably not set standard time) and dipped in xylene prior to cover slipping. Reason given is that the counterstain gets washed out. Wouldn't adjusting the times be a better resolution. I understand residual water could be present and cause long term issues on storage but wanted some other opinions on this process. Diana ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ** The information contained in this message may be privileged and confidential and protected from disclosure. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please notify us immediately by replying to the message and deleting it from your computer. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC lead
What criteria does everyone use to hire for working in your IHC department? I am looking for feedback as to what qualifications are expected for IHC techs as well as qualifications and expectations of the lead in IHC. Also, what title do you have for the lead in IHC, technical specialist, coordinator, lead, etc??? Thanks ahead of time for your information! We are trying to reinvent the position and want to make it more like a community standard (community meaning the world of histology)!! This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC lead
I always looked for a HTL (ASCP) certified and the title was Senior Histotechnologist René J. From: Webb, Dorothy L dorothy.l.w...@healthpartners.com To: 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu Sent: Wednesday, September 12, 2012 2:17 PM Subject: [Histonet] IHC lead What criteria does everyone use to hire for working in your IHC department? I am looking for feedback as to what qualifications are expected for IHC techs as well as qualifications and expectations of the lead in IHC. Also, what title do you have for the lead in IHC, technical specialist, coordinator, lead, etc??? Thanks ahead of time for your information! We are trying to reinvent the position and want to make it more like a community standard (community meaning the world of histology)!! This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: air drying special stain slides rather than
We have routinely dried slides prior to coverslipping. We have found that ethanol or acetone rinsing after staining shortens the drying time (in fact we have used acetone to dehydrate MGG and DiffQuik stained smears, which must be kept away from alcohols, prior to coverslipping). Our automatic coverslipper uses a very runny xylene based mountant so we do not need to rinse in xylene prior to coverslipping. One paper we did describes the detergent de-waxing aspect and a study currently in preparation applies the technique to fungal staining: Henwood A (2012) The application of heated detergent dewaxing and rehydration to immunohistochemistry Biotechnic Histochemistry 87(1): 46-50. It will depend on the staining method used as to whether you can use alcohol, acetone or heat-assisted drying prior to coverslipping, but, dare I say, nearly all stains can be treated thus. Whoops, I forgot about the Oil Red O stains for fats, Oh well I did say nearly all!! Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mayer,Toysha N Sent: Wednesday, 12 September 2012 1:42 AM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] RE: air drying special stain slides rather than Ooh, great question for my students next semester. Your answer is the counterstain, some counterstains may require dehydration after rinsing, or some may not. Adjusting the times of the counterstain is not the issue as much as the solvent of the counterstain. Rene, while I do acknowledge that the xylene may/will cause hazards, we must think of the miscibility of the clearant and the dehydrant, as well as the amount of time involved. The amount of time involved to blot and air dry the slides will affect the TAT for the specimen. 5 min may be ok if you have a small amount of slides, but with a larger number of slides, it will be considerably more than 5. Also Lean methodologies would not apply in that case. With automation, the extreme heat involved with a stain dryer may affect the tissue on the slide. There are some stains that can be blotted, cleared and coverslipped, but using the alcohol to remove excess water and counter stain is better in my opinion. Toysha N. Mayer, MBA, HT (ASCP) Instructor, Education Coordinator Program in Histotechnology School of Health Professions MD Anderson Cancer Center (713) 563-3481 tnma...@mdanderson.org Message: 16 Date: Tue, 11 Sep 2012 10:32:08 -0400 From: Diana McCaig dmcc...@ckha.on.ca Subject: [Histonet] air drying special stain slides rather than dehydrate and clear To: histonet@lists.utsouthwestern.edu Message-ID: dcfd9e6a390e294aaf3a2561cd32e5c417a90...@ckhamail1.ckha.on.ca Content-Type: text/plain; charset=us-ascii I was hoping to get information on why special stains are dehydrated, cleared and mounted vs allowing them to be blotted dry, air dried then coverslip. Every procedure I have ever encountered always indicates to dehydrate and clear but I have heard where some labs are blotting the slides , allowing to air dry (probably not set standard time) and dipped in xylene prior to cover slipping. Reason given is that the counterstain gets washed out. Wouldn't adjusting the times be a better resolution. I understand residual water could be present and cause long term issues on storage but wanted some other opinions on this process. Diana -- Message: 17 Date: Tue, 11 Sep 2012 07:52:05 -0700 (PDT) From: Rene J Buesa rjbu...@yahoo.com Subject: Re: [Histonet] air drying special stain slides rather than dehydrate and clear To: Diana McCaig dmcc...@ckha.on.ca, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: 1347375125.72189.yahoomail...@web121405.mail.ne1.yahoo.com Content-Type: text/plain; charset=iso-8859-1 Diana: The most simple answer to your question is: Because that is the way it has been done for more than 150 years. The second question would be: Is it necessary? and the short answer to this question is: NO!!! As a matter of fact, one of the steps I have developed to totally eliminate xylene from the histology lab refers to the clearing of stained sections, not only special stains (the so called HC and IHC) but the routine as well (the HE). Now, the secret to a successful drying of the stained slides is NOT to let them air dry because that will take not only too much time, but you can never be sure if the section is completely dry and if you add the mounting medium to a not completely dried section, you will have transparency
RE: [Histonet] RE: air drying special stain slides rather than
Yep Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Diana McCaig Sent: Wednesday, 12 September 2012 3:23 AM To: E. Wayne Johnson; Rene J Buesa Cc: histonet@lists.utsouthwestern.edu; Mayer, Toysha N Subject: RE: [Histonet] RE: air drying special stain slides rather than Would this work for auto cover slipping (tape film)if they were set in the xylene reservoir prior to cover slipping? Diana -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of E. Wayne Johnson Sent: September-11-12 1:15 PM To: Rene J Buesa Cc: 'histonet@lists.utsouthwestern.edu'; Mayer, Toysha N Subject: Re: [Histonet] RE: air drying special stain slides rather than I am convinced to give it a try because I also have trouble will the loss of some stains in dehydration. I was concerned that the slides would not clear well after oven dehydration. I will see how it works for me. I can see clearly how going from counterstain to oven will save much hassle with xylene and alcohols as well as not washing out some special stains. I have tried some of the isopropyl alcohol and acetone dehydration called for in some of the stain procedures and it would be great if the slides could just be popped into the oven. What mounting medium are you using? Does it matter? I am a bit worried about penetration of the mountant into the tissue section if there is no xylene in the tissue. Will neutral balsam still work ok? Rene: if you have a link to the paper you talked about on eliminating xylene, I am interested. Xylene is becoming more and more of an issue and a pain for us. EWJohnson Enruikang Ag Tech Beijing. On 9/12/2012 12:01 AM, Rene J Buesa wrote: Toysha: Perhaps you have not oven dried stained slides before, and that explains some of your comments, like: 1- if the stained slides are completely dried, the miscibility you point out is not an issues, because there is nothing to mix with; 2- if you dehydrate → clear the stained sections that will take about 15 minutes per group of up to 25 slides, or even more depending on the protocol used in your automated stainer, but if your group of slides in their rack are placed in an oven at 60ºC for 5 minutes it will just that, 5 minutes reducing the usual TAT for each staining procedure; 3- any oven can accommodate more than 100 stained slides in their racks and the TAT is shortened by oven drying, no matter how many slides you are working with; 4- I really do not know where you can find that extreme heat can affect the tissue sections. All tissue sections are fixed → processed → dried (usually at the same 60ºC before staining) → stained and an additional step at 60ºC to dry before cover-slipping is just that, an additional step at 60ºC 5- The so called Lean technologies do not refer to staining only, they have to do with the whole work-flow and an additional drying step at 60ºC cannot affect in a negative way to the work-flow 6- after staining you will oven dry the sections. I think you should try the method instead. René J. From: Mayer,Toysha Ntnma...@mdanderson.org To: 'histonet@lists.utsouthwestern.edu'histo...@lists.utsouthwestern.ed u Sent: Tuesday, September 11, 2012 11:41 AM Subject: [Histonet] RE: air drying special stain slides rather than Ooh, great question for my students next semester. Your answer is the counterstain, some counterstains may require dehydration after rinsing, or some may not. Adjusting the times of the counterstain is not the issue as much as the solvent of the counterstain. Rene, while I do acknowledge that the xylene may/will cause hazards, we must think of the miscibility of the clearant and the dehydrant, as well as the amount of time involved. The amount of time involved to blot and air dry the slides will affect the TAT for the specimen. 5 min may be ok if you have a small amount of slides, but with a larger number of slides, it will be considerably more than 5. Also Lean methodologies would not apply in that case. With automation, the extreme heat involved with a stain dryer may affect the tissue on the slide. There are some stains that can be blotted, cleared and coverslipped, but using the alcohol to remove excess water and counter stain is better in my opinion. Toysha N. Mayer, MBA, HT (ASCP) Instructor, Education Coordinator Program in Histotechnology School of Health Professions MD Anderson Cancer Center (713) 563-3481 tnma...@mdanderson.org Message:
Re: [Histonet] RE: air drying special stain slides rather than
Interestingly, I showed the results to a couple of colleagues. One response was- The sections will come off! The sections will come off! All that heat! The soap! The sections will come off! I don't think I even want to try That! I'm going to stick with Xylene and Alcohols. Another - Oh, this method is /Very Unusual/. Maybe if the graduate students try to publish a scientific paper and say that they used this method, their papers will be *Rejected* *by the Reviewers*. It's a published method. I have 2 published papers on it right here Oh, so they can cite those methods in their papers. Ok. * E. Wayne Johnson Enruikang Ag Tech Beijing On 9/13/2012 8:41 AM, Tony Henwood (SCHN) wrote: Yep Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Diana McCaig Sent: Wednesday, 12 September 2012 3:23 AM To: E. Wayne Johnson; Rene J Buesa Cc: histonet@lists.utsouthwestern.edu; Mayer, Toysha N Subject: RE: [Histonet] RE: air drying special stain slides rather than Would this work for auto cover slipping (tape film)if they were set in the xylene reservoir prior to cover slipping? Diana -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of E. Wayne Johnson Sent: September-11-12 1:15 PM To: Rene J Buesa Cc: 'histonet@lists.utsouthwestern.edu'; Mayer, Toysha N Subject: Re: [Histonet] RE: air drying special stain slides rather than I am convinced to give it a try because I also have trouble will the loss of some stains in dehydration. I was concerned that the slides would not clear well after oven dehydration. I will see how it works for me. I can see clearly how going from counterstain to oven will save much hassle with xylene and alcohols as well as not washing out some special stains. I have tried some of the isopropyl alcohol and acetone dehydration called for in some of the stain procedures and it would be great if the slides could just be popped into the oven. What mounting medium are you using? Does it matter? I am a bit worried about penetration of the mountant into the tissue section if there is no xylene in the tissue. Will neutral balsam still work ok? Rene: if you have a link to the paper you talked about on eliminating xylene, I am interested. Xylene is becoming more and more of an issue and a pain for us. EWJohnson Enruikang Ag Tech Beijing. On 9/12/2012 12:01 AM, Rene J Buesa wrote: Toysha: Perhaps you have not oven dried stained slides before, and that explains some of your comments, like: 1- if the stained slides are completely dried, the miscibility you point out is not an issues, because there is nothing to mix with; 2- if you dehydrate → clear the stained sections that will take about 15 minutes per group of up to 25 slides, or even more depending on the protocol used in your automated stainer, but if your group of slides in their rack are placed in an oven at 60ºC for 5 minutes it will just that, 5 minutes reducing the usual TAT for each staining procedure; 3- any oven can accommodate more than 100 stained slides in their racks and the TAT is shortened by oven drying, no matter how many slides you are working with; 4- I really do not know where you can find that extreme heat can affect the tissue sections. All tissue sections are fixed → processed → dried (usually at the same 60ºC before staining) → stained and an additional step at 60ºC to dry before cover-slipping is just that, an additional step at 60ºC 5- The so called Lean technologies do not refer to staining only, they have to do with the whole work-flow and an additional drying step at 60ºC cannot affect in a negative way to the work-flow 6- after staining you will oven dry the sections. I think you should try the method instead. René J. From: Mayer,Toysha Ntnma...@mdanderson.org To: 'histonet@lists.utsouthwestern.edu'histo...@lists.utsouthwestern.ed u Sent: Tuesday, September 11, 2012 11:41 AM Subject: [Histonet] RE: air drying special stain slides rather than Ooh, great question for my students next semester. Your answer is the counterstain, some counterstains may require dehydration after rinsing, or some may not. Adjusting the times of the counterstain is not the issue as much as the solvent of the counterstain. Rene, while I do acknowledge that the xylene may/will cause hazards, we must think of the miscibility of the clearant and the dehydrant, as well as the amount of time involved. The amount of time involved to blot and air dry the slides will affect the TAT for the specimen. 5 min may be
