Re: [Histonet] VIP issue
Gudrun: I have used/recommended for 10 years now graded isopropyl (2-propanol) alcohol to dehydrate and before paraffin ONLY and when I went to Australia in 2011 found out that they had been using the same protocol for years. You just grade 2-propanol in the same way you grade ethanol (60%→80%→90%→100%) and continue DIRECTLY to paraffin. Usually I dedicate one "hot" (paraffin) VIP station with a 1:1 of 2-propanol + paraffin mixture to facilitate the infiltration or (better yet) one "hot" station with a 1:1 mixture of 2-propanol + pure mineral oil (= paraffin of low molecular weight) and the infiltration is really excellent. In this way you will be able to eliminate BOTH xylene and any other "friendly" clearing agent (ALL are toxic lat different evels). 2-propanol is NOT toxic AT ALL! I have ALSO stopped using xylene to de-wax sections before staining and substituted it with a 2% aq. sol. of dishwashing soap and go to water → staining after (I can send you the detailed protocol / publications). AFTER staining I just rinse the stained slides in 2-propalo → shake it well → to dry oven at 60ºC for 10 minutes → coverslip. My lab was TOTALLY xylene free, and you can do the same thing. If you are hesitant just TRY these methods with only few blocks / sections / stained sections and convince yourself. Best regards René On Friday, October 12, 2018 1:13 PM, Gudrun Lang via Histonet wrote: Thank you for all the good hints and advices. It has to be a reagens-issue, because the second instrument was also concerned. But we had no similar problems for decades, although we use 96% as cleaning-ethanol after ShellSol. We change the reagenses after 5 runs, once a week. We soon are forced to end using ShellSol, because the company stops the production. We don't like to switch to xylene. Which xylen-substitutes are recommended for the VIP besides of Tissue-Clear (Sakura)? Best wishes Gudrun -Ursprüngliche Nachricht- Von: Gudrun Lang via Histonet [mailto:histonet@lists.utsouthwestern.edu] Gesendet: Donnerstag, 11. Oktober 2018 18:58 An: histonet@lists.utsouthwestern.edu Betreff: [Histonet] VIP issue Dear all! I have a question for those, who are familiar with the VIPs from Sakura. Last time we changed the reagenses the cleaning-ethanol (96%) was very milky and even was full of many small particles. It was a paraffin-soup. What is the cause for such a case? We have been using the organic solvent (ShellSol) for decades as xylensubstitute. Maybe the quality has suffered and the ability of solving paraffin has decreased. But are there other explanations? Maybe a malfunction of the instrument? Thanks in advance Gudrun Lang ___ 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 mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] VIP issue
Thank you for all the good hints and advices. It has to be a reagens-issue, because the second instrument was also concerned. But we had no similar problems for decades, although we use 96% as cleaning-ethanol after ShellSol. We change the reagenses after 5 runs, once a week. We soon are forced to end using ShellSol, because the company stops the production. We don't like to switch to xylene. Which xylen-substitutes are recommended for the VIP besides of Tissue-Clear (Sakura)? Best wishes Gudrun -Ursprüngliche Nachricht- Von: Gudrun Lang via Histonet [mailto:histonet@lists.utsouthwestern.edu] Gesendet: Donnerstag, 11. Oktober 2018 18:58 An: histonet@lists.utsouthwestern.edu Betreff: [Histonet] VIP issue Dear all! I have a question for those, who are familiar with the VIPs from Sakura. Last time we changed the reagenses the cleaning-ethanol (96%) was very milky and even was full of many small particles. It was a paraffin-soup. What is the cause for such a case? We have been using the organic solvent (ShellSol) for decades as xylensubstitute. Maybe the quality has suffered and the ability of solving paraffin has decreased. But are there other explanations? Maybe a malfunction of the instrument? Thanks in advance Gudrun Lang ___ 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] VIP issue
Gudrun- Assuming this is a new issue with a recent reagent change, I suspect that the alcohol used for the clean cycle was not 100% (maybe 95%?). The ability of your alcohol to hold the paraffin in solution is lost as the percentage of water increases. I also expect your solvent was mostly saturated with paraffin. Try changing your clean cycle reagents and see if the situation improves. I would do this before processing tissue again on this unit. I hope this helps. Joe Saby On Thursday, October 11, 2018, 1:48:54 PM EDT, Gudrun Lang via Histonet wrote: Dear all! I have a question for those, who are familiar with the VIPs from Sakura. Last time we changed the reagenses the cleaning-ethanol (96%) was very milky and even was full of many small particles. It was a paraffin-soup. What is the cause for such a case? We have been using the organic solvent (ShellSol) for decades as xylensubstitute. Maybe the quality has suffered and the ability of solving paraffin has decreased. But are there other explanations? Maybe a malfunction of the instrument? Thanks in advance Gudrun Lang ___ 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] VIP issue
Either: 1) the ShellSol was over-used and could no longer clean/dissolve the paraffin so the remaining paraffin ended up in the alcohol 2) There was water in the cleaning alcohol (we always use 100% alcohol - do you always use 96%? 3) the cleaning ShellSol and cleaning alcohols were switched 4) there was a machine malfunction Good Luck! Laurie Redmond -Original Message- From: Gudrun Lang via Histonet To: histonet Sent: Thu, Oct 11, 2018 10:58 am Subject: [Histonet] VIP issue Dear all! I have a question for those, who are familiar with the VIPs from Sakura. Last time we changed the reagenses the cleaning-ethanol (96%) was very milky and even was full of many small particles. It was a paraffin-soup. What is the cause for such a case? We have been using the organic solvent (ShellSol) for decades as xylensubstitute. Maybe the quality has suffered and the ability of solving paraffin has decreased. But are there other explanations? Maybe a malfunction of the instrument? Thanks in advance Gudrun Lang ___ 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] VIP issue
Dear all! I have a question for those, who are familiar with the VIPs from Sakura. Last time we changed the reagenses the cleaning-ethanol (96%) was very milky and even was full of many small particles. It was a paraffin-soup. What is the cause for such a case? We have been using the organic solvent (ShellSol) for decades as xylensubstitute. Maybe the quality has suffered and the ability of solving paraffin has decreased. But are there other explanations? Maybe a malfunction of the instrument? Thanks in advance Gudrun Lang ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
