RE: [Histonet] DRGs histogel
I think dehydration during processing can effect the outcome with histogel. I use it frequently to help orient small samples with very good results almost always. One time, I was trying to arrange several samples in the same block and the histogel got too cold and set up when I was partly finished. I put it all in our oven hoping to remelt it. When I took it out, it had become a hard thin sheet, sort of like dried paint or glue. The only thing I could think of that had happened, was that since it was in a shape with a lot of surface area, it had dried out instead of melting. I wanted to save the sample that was in it, so I added some water. Thankfully, the histogel became gel like again and I was able to find the sample which I transfered to fresh histogel. The reconstituted histogel seemed to be the same as regular histogel, excep it lost its pink color. The samples also were ok. I've never had any real problems cutting histogel (and hope I don't), but if I get one that is a little crumbly, I soak it extra long and that helps. I think that maybe when there are inconsistent results with histogel, maybe something happens during processing so it becomes dried out or overly dehydrated. I have used it both out of formalin and out of 70%. I've also embedded it both wet and dry without problems. Sometimes it is whiter than others. Karen Doty Date: Mon, 22 Feb 2010 09:35:10 -0800 From: dunat...@sbcglobal.net To: port...@msu.edu; algra...@email.arizona.edu; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] DRGs CC: I also think that it is strange of the way Histogel processes. I have posted on the Histonet previously about this exact problem. I worked with Jennifer Hofecker when she was at Vanderbilt U.(sent her my Histogel and she sent me hers) and ended up with perfectly processed Histogel blocks at our facility and hers. I processed a couple of blocks last week and they were just terrible. No change in the processing schedule, or the way Histogel was liquefied (placed in hot water that was heated in the microwave). Prior to the last two blocks, I must have processed at least a dozen blocks without any problems. There was an incident where I placed two histogels in the same cassete. One processed beautifuly and the other was all shrunken and dried up. I do not liquefy the entire tube, rather I scoop out the approximate amount that I need and transfer to another tube to heat up. If there is anyone out there in Histoland that has not had any issues with the Histogel, can you please post your procedure on liquefying the Histogel, method of cooling/solidifying and processing schedule? The only thing that I do that is not exact is I do not know the temp of my hot water when i place the Histogel to liquefy. I basically have to wait several minutes for the gel to melt and I use it immediately. Any new information or solution from anyone, would be greatly appreciated. Thank you Dusko Trajkovic From: Amy Porter port...@msu.edu To: Andrea Grantham algra...@email.arizona.edu; HISTONET histonet@lists.utsouthwestern.edu Sent: Mon, February 22, 2010 9:01:22 AM Subject: RE: [Histonet] DRGs I think it is strange that we are all doing similar techniques and wind up with different outcomes using the histogel. I would be curious how many of us are using the equipment sold with the histogel for warming and cooling opposed to any of us who don't. we did not purchase the equipment and I wonder sometimes if warming the histogel using other means causes some type of breakdown / and do any of you repeatedly reheat the same tube once it has been warmed and resolidified?? -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea Grantham Sent: Monday, February 22, 2010 9:41 AM To: HISTONET Subject: Re: [Histonet] DRGs Importance: High Hi Carol, I have used histogel for these kinds of samples and also other small, thin tissues like insect antennae and insect GI tracts and midguts. Since I get all my projects already fixed in whatever fixative the investigator chooses, rinsed and placed in 70% ETOH the histogel never touches formalin. I don't use formalin on my processor but start in 70%. I've never had a problem with the histogel. We just put the sample in the histogel flat and stand it up (turn 90°) when embedding in paraffin. I use tissue prep for embedding. If you don't want to use histogel you could try to put the drg's on GN Metricel membrane disc filters. We do this with a lot of the samples I receive, actually I have the investigators or their techs do this. The tissue sticks to the membrane and orientation is a dream. The membrane presents no problem when sectioning. You can get it from VWR. Andi Andrea Grantham, HT (ASCP) Senior Research
Re: [Histonet] DRGs
Hi Carol, I have used histogel for these kinds of samples and also other small, thin tissues like insect antennae and insect GI tracts and midguts. Since I get all my projects already fixed in whatever fixative the investigator chooses, rinsed and placed in 70% ETOH the histogel never touches formalin. I don't use formalin on my processor but start in 70%. I've never had a problem with the histogel. We just put the sample in the histogel flat and stand it up (turn 90°) when embedding in paraffin. I use tissue prep for embedding. If you don't want to use histogel you could try to put the drg's on GN Metricel membrane disc filters. We do this with a lot of the samples I receive, actually I have the investigators or their techs do this. The tissue sticks to the membrane and orientation is a dream. The membrane presents no problem when sectioning. You can get it from VWR. Andi Andrea Grantham, HT (ASCP) Senior Research Specialist University of Arizona Cell Biology and Anatomy Histology Service Laboratory P.O.Box 245044 Tucson, AZ 85724 algra...@email.arizona.edu Tel: 520.626.4415 Fax: 520.626.2097 happy slicing and dicing and may all your stains work perfectly - Paula Sicurello P Please consider the environment before printing this email. On Feb 18, 2010, at 11:23 AM, Barone, Carol wrote: Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx- ASAP! cbar...@nemours.org ___ 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] DRGs
I think it is strange that we are all doing similar techniques and wind up with different outcomes using the histogel. I would be curious how many of us are using the equipment sold with the histogel for warming and cooling opposed to any of us who don't. we did not purchase the equipment and I wonder sometimes if warming the histogel using other means causes some type of breakdown / and do any of you repeatedly reheat the same tube once it has been warmed and resolidified?? -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea Grantham Sent: Monday, February 22, 2010 9:41 AM To: HISTONET Subject: Re: [Histonet] DRGs Importance: High Hi Carol, I have used histogel for these kinds of samples and also other small, thin tissues like insect antennae and insect GI tracts and midguts. Since I get all my projects already fixed in whatever fixative the investigator chooses, rinsed and placed in 70% ETOH the histogel never touches formalin. I don't use formalin on my processor but start in 70%. I've never had a problem with the histogel. We just put the sample in the histogel flat and stand it up (turn 90°) when embedding in paraffin. I use tissue prep for embedding. If you don't want to use histogel you could try to put the drg's on GN Metricel membrane disc filters. We do this with a lot of the samples I receive, actually I have the investigators or their techs do this. The tissue sticks to the membrane and orientation is a dream. The membrane presents no problem when sectioning. You can get it from VWR. Andi Andrea Grantham, HT (ASCP) Senior Research Specialist University of Arizona Cell Biology and Anatomy Histology Service Laboratory P.O.Box 245044 Tucson, AZ 85724 algra...@email.arizona.edu Tel: 520.626.4415 Fax: 520.626.2097 happy slicing and dicing and may all your stains work perfectly - Paula Sicurello P Please consider the environment before printing this email. On Feb 18, 2010, at 11:23 AM, Barone, Carol wrote: Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx- ASAP! cbar...@nemours.org ___ 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] DRGs
I also think that it is strange of the way Histogel processes. I have posted on the Histonet previously about this exact problem. I worked with Jennifer Hofecker when she was at Vanderbilt U.(sent her my Histogel and she sent me hers) and ended up with perfectly processed Histogel blocks at our facility and hers. I processed a couple of blocks last week and they were just terrible. No change in the processing schedule, or the way Histogel was liquefied (placed in hot water that was heated in the microwave). Prior to the last two blocks, I must have processed at least a dozen blocks without any problems. There was an incident where I placed two histogels in the same cassete. One processed beautifuly and the other was all shrunken and dried up. I do not liquefy the entire tube, rather I scoop out the approximate amount that I need and transfer to another tube to heat up. If there is anyone out there in Histoland that has not had any issues with the Histogel, can you please post your procedure on liquefying the Histogel, method of cooling/solidifying and processing schedule? The only thing that I do that is not exact is I do not know the temp of my hot water when i place the Histogel to liquefy. I basically have to wait several minutes for the gel to melt and I use it immediately. Any new information or solution from anyone, would be greatly appreciated. Thank you Dusko Trajkovic From: Amy Porter port...@msu.edu To: Andrea Grantham algra...@email.arizona.edu; HISTONET histonet@lists.utsouthwestern.edu Sent: Mon, February 22, 2010 9:01:22 AM Subject: RE: [Histonet] DRGs I think it is strange that we are all doing similar techniques and wind up with different outcomes using the histogel. I would be curious how many of us are using the equipment sold with the histogel for warming and cooling opposed to any of us who don't. we did not purchase the equipment and I wonder sometimes if warming the histogel using other means causes some type of breakdown / and do any of you repeatedly reheat the same tube once it has been warmed and resolidified?? -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea Grantham Sent: Monday, February 22, 2010 9:41 AM To: HISTONET Subject: Re: [Histonet] DRGs Importance: High Hi Carol, I have used histogel for these kinds of samples and also other small, thin tissues like insect antennae and insect GI tracts and midguts. Since I get all my projects already fixed in whatever fixative the investigator chooses, rinsed and placed in 70% ETOH the histogel never touches formalin. I don't use formalin on my processor but start in 70%. I've never had a problem with the histogel. We just put the sample in the histogel flat and stand it up (turn 90°) when embedding in paraffin. I use tissue prep for embedding. If you don't want to use histogel you could try to put the drg's on GN Metricel membrane disc filters. We do this with a lot of the samples I receive, actually I have the investigators or their techs do this. The tissue sticks to the membrane and orientation is a dream. The membrane presents no problem when sectioning. You can get it from VWR. Andi Andrea Grantham, HT (ASCP) Senior Research Specialist University of Arizona Cell Biology and Anatomy Histology Service Laboratory P.O.Box 245044 Tucson, AZ 85724 algra...@email.arizona.edu Tel: 520.626.4415 Fax: 520.626.2097 happy slicing and dicing and may all your stains work perfectly - Paula Sicurello P Please consider the environment before printing this email. On Feb 18, 2010, at 11:23 AM, Barone, Carol wrote: Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx- ASAP! cbar...@nemours.org ___ 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
Re: [Histonet] DRGs and Histogel
I have had the exact same results, one piece processing well and another in the same cassette shrinking and hardening, when using 1.5-2% low melting point agarose (NuSieve) instead of Histogel. I was thinking of changing to Histogel, but then this thread started last summer and now I see issues continue. I would love to know the best way to do this also! Esther Peters, Ph.D. George Mason University dusko trajkovic wrote: I also think that it is strange of the way Histogel processes. I have posted on the Histonet previously about this exact problem. I worked with Jennifer Hofec ker when she was at Vanderbilt U.(sent her my Histogel and she sent me hers) an d ended up with perfectly processed Histogel blocks at our facility and hers. I processed a couple of blocks last week and they were just terrible. No change in the processing schedule, or the way Histogel was liquefied (placed in hot wa ter that was heated in the microwave). Prior to the last two blocks, I must hav e processed at least a dozen blocks without any problems. There was an incident where I placed two histogels in the same cassete. One processed beautifuly and the other was all shrunken and dried up. I do not liquefy the entire tube, rather I scoop out the approximate amount tha t I need and transfer to another tube to heat up. If there is anyone out there in Histoland that has not had any issues with the Histogel, can you please post your procedure on liquefying the Histogel, method of cooling/solidifying and p rocessing schedule? The only thing that I do that is not exact is I do not know the temp of my hot water when i place the Histogel to liquefy. I basically hav e to wait several minutes for the gel to melt and I use it immediately. Any new information or solution from anyone, would be greatly appreciated. Thank you Dusko Trajkovic From: Amy Porter [1]port...@msu.edu To: Andrea Grantham [2]algra...@email.arizona.edu; HISTONET [3]histo...@list s.utsouthwestern.edu Sent: Mon, February 22, 2010 9:01:22 AM Subject: RE: [Histonet] DRGs I think it is strange that we are all doing similar techniques and wind up with different outcomes using the histogel. I would be curious how many of us are using the equipment sold with the histogel for warming and cooling opposed to any of us who don't. we did not purchase the equipment and I wonder sometimes if warming the histogel using other means causes some type of breakdown / and do any of you repeatedly reheat the same tube once it has been warmed and resolidified?? -Original Message- From: [4]histonet-boun...@lists.utsouthwestern.edu [[5]mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea Grantham Sent: Monday, February 22, 2010 9:41 AM To: HISTONET Subject: Re: [Histonet] DRGs Importance: High Hi Carol, I have used histogel for these kinds of samples and also other small, thin tissues like insect antennae and insect GI tracts and midguts. Since I get all my projects already fixed in whatever fixative the investigator chooses, rinsed and placed in 70% ETOH the histogel never touches formalin. I don't use formalin on my processor but start in 70%. I've never had a problem with the histogel. We just put the sample in the histogel flat and stand it up (turn 90°) when embedding in paraffin. I use tissue prep for embedding. If you don't want to use histogel you could try to put the drg's on GN Metricel membrane disc filters. We do this with a lot of the samples I receive, actually I have the investigators or their techs do this. The tissue sticks to the membrane and orientation is a dream. The membrane presents no problem when sectioning. You can get it from VWR. Andi Andrea Grantham, HT (ASCP) Senior Research Specialist University of Arizona Cell Biology and Anatomy Histology Service Laboratory P.O.Box 245044 Tucson, AZ 85724 [6]algra...@email.arizona.edu Tel: 520.626.4415Fax: 520.626.2097 happy slicing and dicing and may all your stains work perfectly - Paula Sicurello P Please consider the environment before printing this email. On Feb 18, 2010, at 11:23 AM, Barone, Carol wrote: Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx
RE: [Histonet] DRGs
Carol, I have had problems with cutting samples setup in histogel before but I can't figure out why, the problem is inconsistent, sometimes the samples are fine and other times it acts like the histogel prevented the infiltration of processing reagents into the tissue. I have even cut the histogel away (it has done it's job of keeping the small tissue oriented) and reprocessed the tissue with some good results. Melt the block and cut the histogel away with a razor blade, it just surrounds the tissue and does not really infiltrate it, then gently squeeze the melted paraffin out while on a hot embedding plate between paper towel, no need to remove paraffin with xylene, just re cassette the tissue keeping track of the orientation and put it back thru the processor. Some theories I have for why histogel embedded samples cut well sometimes and not others include: 1. Maybe the histogel got too cold during the setup process, I try to make sure the histogel is completely liquid (warmed) when I put it in the mold with the tissue and then let them cool together, I think sometimes if I am preparing a bunch of samples the last ones start to setup before I put the tissue in the mold with the histogel, but I have not done a scientific study of this. 2. I think that it might be better to go into formalin rather than 70% alcohol after preparing the histogel embedded block as the alcohol may harden the histogel too much too quickly preventing infiltration? Again, just a theory not tested in a controlled way?? Sometimes my samples are already in alcohol and sometimes they are in formalin so I have been putting them back into what they came in. Regards, Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. Ste.215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 www.ihctech.net www.ihcrg.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Barone, Carol Sent: Thursday, February 18, 2010 11:24 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] DRGs Importance: High Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx- ASAP! cbar...@nemours.org ___ 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] DRGs
Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx- ASAP! cbar...@nemours.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] DRGs
We normally do CG's and DRG's in Frozen format as well and have had difficulty working with histogel and low percetages of ethanol. The histogel doesn't really bond with the samples very well when taken from an low % ethanol for us. Amy S. Porter, HT (ASCP) QIHC Investigative HistoPathology Laboratory - Supervisor 2201 Biomedical Physical Sciences Bldg. Rm #2133 East Lansing, MI 48824-3320 Phone: (517) 884-5026 Fax: (517) 432-1368 Email: port...@msu.edu Web: www.humanpathology.msu.edu - Original Message - From: Barone, Carol cbar...@nemours.org To: histonet@lists.utsouthwestern.edu Sent: Thursday, February 18, 2010 1:23 PM Subject: [Histonet] DRGs Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx- ASAP! cbar...@nemours.org ___ 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
