RE: [Histonet] prevent wrinkles when cutting
Rachel We also run multiple antibodies under multiple parameters and there is no need or reason to cut in one ribbon that many sections. You will still be able collect somewhat serial or the sections that you need with multiple ribbons. You are only able to fit on the waterbath in one ribbon (depending upon the size base mold that you used ) anywhere from 6 to 15 sections. You can place ribbons side by side on the waterbath but for me personally I find it’s a bit more difficult to pick the sections up once you have placed multiple long ribbons on the waterbath. I prefer working with one ribbon at a time, two tops when collecting multiple sections, but that’s just me. We work with animal tissue and tissue that may not have been processed in our lab so we do soak all of the blocks. The other thing you need to consider is that when we section a paraffin block the section thickness is not exactly consistent, we are essentially cutting a wedge as the block warms the sections becomes thicker, so if we can control the number of sections we collect in one ribbon, place the block back on ice before we collect the next ribbon, we can then maintain section thickness better. I would think that as you section 30 +sections in one ribbon the block is warming up and the sections are becoming thicker. Section thickness is extremely important when comparing IHC staining results, staining intensity will change in a 1 micron difference in section thickness. Just my two cents - and I do have images and staining intensity calculations with respects to section thickness. This is something that we have looked at recently. Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell l...@premierlab.com www.premierlab.com March 10, 2014 is Histotechnology Professionals Day Ship to Address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rachel M Gonzalez Sent: Monday, April 20, 2015 3:05 PM To: Joelle Weaver Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] prevent wrinkles when cutting Hi The long ribbons are for RD and QA purposes. Often we test under multiple conditions 1-4 with multiple antibodies to the same tissue or multiple tissue to optimize reagents. It quickly adds up. Rachel On Mon, Apr 20, 2015 at 3:28 PM, Joelle Weaver joellewea...@hotmail.com wrote: Why are you cutting such a long ribbon? You usually only a need a series of 3-4 sections even for ribbon cutting. Might be easier to control if you don't try to move such a long ribbon to the waterbath. Drag the shorter ribbon towards you on the waterbath. Make sure the water is not too cool. Face the block to full face but superficial, chill on ice for some moisture, take sections while the block is still very cold. Use a slow, steady, smooth stroke if doing manual cutting. Make sure your embedding works well for the way you orient the block in the holder. Angles work well for many tissues that are prone to wrinkling. Its mostly just practice though. The more you cut, the easier it becomes and usually the better you get at it. Joelle Weaver MAOM, HTL (ASCP) QIHC Date: Mon, 20 Apr 2015 19:06:17 +0200 From: j.benavi...@eae.csic.es To: histonet@lists.utsouthwestern.edu Subject: Re: FW: [Histonet] prevent wrinkles when cutting Hi there, I´m curious about the soaking thing. We have never done it in our lab. Which is the purpose to do it? Than, after facing the blocks, we chill them in a cold plate so, if wanting to do the soaking , when should we? I guess before placing them on the cold plate, but that may cause a bit of ice formation? Thanks a lot for your help Julio On 20/04/2015 18:40, Grantham, Andrea L - (algranth) wrote: Rachel, First off, are you chilling and soaking the blocks after you face them? Do that and see if there is a difference. Don't try to get many sections to your ribbons. Shoot for a smaller ribbon (5-6) sections that are good. Cut slowly but consistently. What microtome are you using? Are you using disposable blades and are they sharp? Don't expect them to cut well if you use the same blade to face the blocks. If you aren't using disposables, get some! They will make your life easier. You might try to find a histotech at a local hospital lab who might be able to give you a hands-on lesson. Don't despair! We all sat down at our microtomes those first times and suffered trying to get perfect sections. It takes practice. You might make some blank blocks or blocks with tissue you can spare to practice your cutting techniques. I used to do this with my students and it really helped them. Good luck! Andi G
Re: [Histonet] prevent wrinkles when cutting
Hi The long ribbons are for RD and QA purposes. Often we test under multiple conditions 1-4 with multiple antibodies to the same tissue or multiple tissue to optimize reagents. It quickly adds up. Rachel On Mon, Apr 20, 2015 at 3:28 PM, Joelle Weaver joellewea...@hotmail.com wrote: Why are you cutting such a long ribbon? You usually only a need a series of 3-4 sections even for ribbon cutting. Might be easier to control if you don't try to move such a long ribbon to the waterbath. Drag the shorter ribbon towards you on the waterbath. Make sure the water is not too cool. Face the block to full face but superficial, chill on ice for some moisture, take sections while the block is still very cold. Use a slow, steady, smooth stroke if doing manual cutting. Make sure your embedding works well for the way you orient the block in the holder. Angles work well for many tissues that are prone to wrinkling. Its mostly just practice though. The more you cut, the easier it becomes and usually the better you get at it. Joelle Weaver MAOM, HTL (ASCP) QIHC Date: Mon, 20 Apr 2015 19:06:17 +0200 From: j.benavi...@eae.csic.es To: histonet@lists.utsouthwestern.edu Subject: Re: FW: [Histonet] prevent wrinkles when cutting Hi there, I´m curious about the soaking thing. We have never done it in our lab. Which is the purpose to do it? Than, after facing the blocks, we chill them in a cold plate so, if wanting to do the soaking , when should we? I guess before placing them on the cold plate, but that may cause a bit of ice formation? Thanks a lot for your help Julio On 20/04/2015 18:40, Grantham, Andrea L - (algranth) wrote: Rachel, First off, are you chilling and soaking the blocks after you face them? Do that and see if there is a difference. Don't try to get many sections to your ribbons. Shoot for a smaller ribbon (5-6) sections that are good. Cut slowly but consistently. What microtome are you using? Are you using disposable blades and are they sharp? Don't expect them to cut well if you use the same blade to face the blocks. If you aren't using disposables, get some! They will make your life easier. You might try to find a histotech at a local hospital lab who might be able to give you a hands-on lesson. Don't despair! We all sat down at our microtomes those first times and suffered trying to get perfect sections. It takes practice. You might make some blank blocks or blocks with tissue you can spare to practice your cutting techniques. I used to do this with my students and it really helped them. Good luck! Andi G. From: histonet-boun...@lists.utsouthwestern.edu [ histonet-boun...@lists.utsouthwestern.edu] on behalf of Rachel M Gonzalez [rac...@gbi-inc.com] Sent: Monday, April 20, 2015 9:07 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] prevent wrinkles when cutting Hi Thursday was the first time I ever used a microtome I move to a lab that does not have someone dedicated to cutting. I already miss her. I have no problems getting ribbons of 10-30 sections long but the pieces are half the size of the original block. I am guessing they are wrinkling. What am I doing wrong? Thanks Rachel Senior Scientist ___ 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 ___ 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] prevent wrinkles when cutting
If you process properly, no over dehydration, there is no reason to soak. Keeping block cool will help in cutting. Sharp, clean blade a must. Correct water bath temp and time are the most critical factors, after cutting, to produce a wrinkle free section. Sent from my iPhone On Apr 20, 2015, at 10:12 AM, Julio Benavides j.benavi...@eae.csic.es wrote: Hi there, I´m curious about the soaking thing. We have never done it in our lab. Which is the purpose to do it? Than, after facing the blocks, we chill them in a cold plate so, if wanting to do the soaking , when should we? I guess before placing them on the cold plate, but that may cause a bit of ice formation? Thanks a lot for your help Julio On 20/04/2015 18:40, Grantham, Andrea L - (algranth) wrote: Rachel, First off, are you chilling and soaking the blocks after you face them? Do that and see if there is a difference. Don't try to get many sections to your ribbons. Shoot for a smaller ribbon (5-6) sections that are good. Cut slowly but consistently. What microtome are you using? Are you using disposable blades and are they sharp? Don't expect them to cut well if you use the same blade to face the blocks. If you aren't using disposables, get some! They will make your life easier. You might try to find a histotech at a local hospital lab who might be able to give you a hands-on lesson. Don't despair! We all sat down at our microtomes those first times and suffered trying to get perfect sections. It takes practice. You might make some blank blocks or blocks with tissue you can spare to practice your cutting techniques. I used to do this with my students and it really helped them. Good luck! Andi G. From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Rachel M Gonzalez [rac...@gbi-inc.com] Sent: Monday, April 20, 2015 9:07 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] prevent wrinkles when cutting Hi Thursday was the first time I ever used a microtome I move to a lab that does not have someone dedicated to cutting. I already miss her. I have no problems getting ribbons of 10-30 sections long but the pieces are half the size of the original block. I am guessing they are wrinkling. What am I doing wrong? Thanks Rachel Senior Scientist ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] prevent wrinkles when cutting
Check you blade angle. Keep your blocks cold, change your blade often and just be patient. Tom Podawiltz HT (ASCP) AP Section Head LRGHealthcare -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rachel M Gonzalez Sent: Monday, April 20, 2015 12:07 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] prevent wrinkles when cutting Hi Thursday was the first time I ever used a microtome I move to a lab that does not have someone dedicated to cutting. I already miss her. I have no problems getting ribbons of 10-30 sections long but the pieces are half the size of the original block. I am guessing they are wrinkling. What am I doing wrong? Thanks Rachel Senior Scientist ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet THIS MESSAGE IS CONFIDENTIAL. This e-mail message and any attachments are proprietary and confidential information intended only for the use of the recipient(s) named above. If you are not the intended recipient, you may not print,distribute, or copy this message or any attachments. If you have received this communication in error, please notify the sender by return e-mail and delete this message and any attachments from your computer. Any views or opinions expressed are solely those of the author and do not necessarily represent those of LRGHealthcare. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] prevent wrinkles when cutting
First make sure your block is cold. Second, some models of microtome's and blades don't cut well without dulling the blade a bit.(newer models don't seem to need it.) Lastly some processors don’t fix as well as others you may need to face the blocks in and soak them. Again some places soak in ice water alone, some use fabric softener on the ice and some use ammonia water. (that one is stinky). Some places do soaking at all. I now have a really old microtome and have to blow or breath on the paraffin lightly as I'm cutting, an old technique but effective. Sorry its going to be a lot of trial and error. Maybe you can call the previous tech on how they did it there. Anne -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rachel M Gonzalez Sent: Monday, April 20, 2015 9:07 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] prevent wrinkles when cutting Hi Thursday was the first time I ever used a microtome I move to a lab that does not have someone dedicated to cutting. I already miss her. I have no problems getting ribbons of 10-30 sections long but the pieces are half the size of the original block. I am guessing they are wrinkling. What am I doing wrong? Thanks Rachel Senior Scientist ___ 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] prevent wrinkles when cutting
How thick are you cutting? Is the block cold? Are you using disposable knives? Are you moving the knife when you start sectioning? How fast are you attempting to cut? What kind of tissue are you cutting? Do you know which paraffin is being used? There are many reasons for wrinkles and these are few of the questions we need to answer first. Pam Marcum -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rachel M Gonzalez Sent: Monday, April 20, 2015 11:07 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] prevent wrinkles when cutting Hi Thursday was the first time I ever used a microtome I move to a lab that does not have someone dedicated to cutting. I already miss her. I have no problems getting ribbons of 10-30 sections long but the pieces are half the size of the original block. I am guessing they are wrinkling. What am I doing wrong? Thanks Rachel Senior Scientist ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Confidentiality Notice: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] prevent wrinkles when cutting
Why are you cutting such a long ribbon? You usually only a need a series of 3-4 sections even for ribbon cutting. Might be easier to control if you don't try to move such a long ribbon to the waterbath. Drag the shorter ribbon towards you on the waterbath. Make sure the water is not too cool. Face the block to full face but superficial, chill on ice for some moisture, take sections while the block is still very cold. Use a slow, steady, smooth stroke if doing manual cutting. Make sure your embedding works well for the way you orient the block in the holder. Angles work well for many tissues that are prone to wrinkling. Its mostly just practice though. The more you cut, the easier it becomes and usually the better you get at it. Joelle Weaver MAOM, HTL (ASCP) QIHC Date: Mon, 20 Apr 2015 19:06:17 +0200 From: j.benavi...@eae.csic.es To: histonet@lists.utsouthwestern.edu Subject: Re: FW: [Histonet] prevent wrinkles when cutting Hi there, I´m curious about the soaking thing. We have never done it in our lab. Which is the purpose to do it? Than, after facing the blocks, we chill them in a cold plate so, if wanting to do the soaking , when should we? I guess before placing them on the cold plate, but that may cause a bit of ice formation? Thanks a lot for your help Julio On 20/04/2015 18:40, Grantham, Andrea L - (algranth) wrote: Rachel, First off, are you chilling and soaking the blocks after you face them? Do that and see if there is a difference. Don't try to get many sections to your ribbons. Shoot for a smaller ribbon (5-6) sections that are good. Cut slowly but consistently. What microtome are you using? Are you using disposable blades and are they sharp? Don't expect them to cut well if you use the same blade to face the blocks. If you aren't using disposables, get some! They will make your life easier. You might try to find a histotech at a local hospital lab who might be able to give you a hands-on lesson. Don't despair! We all sat down at our microtomes those first times and suffered trying to get perfect sections. It takes practice. You might make some blank blocks or blocks with tissue you can spare to practice your cutting techniques. I used to do this with my students and it really helped them. Good luck! Andi G. From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Rachel M Gonzalez [rac...@gbi-inc.com] Sent: Monday, April 20, 2015 9:07 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] prevent wrinkles when cutting Hi Thursday was the first time I ever used a microtome I move to a lab that does not have someone dedicated to cutting. I already miss her. I have no problems getting ribbons of 10-30 sections long but the pieces are half the size of the original block. I am guessing they are wrinkling. What am I doing wrong? Thanks Rachel Senior Scientist ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] prevent wrinkles when cutting
The one question we have not asked is; are you cutting serial sections for these blocks? If you are are you laying them out on a board and not a waterbath? I have done that with neuropath sections in research for whole rat and monkey brains. Then your ribbon length would make sense. Generally the longer the ribbon you have to handle the more likely you are to lose sections or have it fold over on its self. When you are just learning mastering shorter ribbons is easier. If you are going to a waterbath you can crowd the ribbon on the bath and it had no place to gently stretch out. Pam Marcum - Original Message - From: Joelle Weaver joellewea...@hotmail.com To: Julio Benavides j.benavi...@eae.csic.es, Histonet histonet@lists.utsouthwestern.edu Sent: Monday, April 20, 2015 2:28:47 PM Subject: RE: [Histonet] prevent wrinkles when cutting Why are you cutting such a long ribbon? You usually only a need a series of 3-4 sections even for ribbon cutting. Might be easier to control if you don't try to move such a long ribbon to the waterbath. Drag the shorter ribbon towards you on the waterbath. Make sure the water is not too cool. Face the block to full face but superficial, chill on ice for some moisture, take sections while the block is still very cold. Use a slow, steady, smooth stroke if doing manual cutting. Make sure your embedding works well for the way you orient the block in the holder. Angles work well for many tissues that are prone to wrinkling. Its mostly just practice though. The more you cut, the easier it becomes and usually the better you get at it. Joelle Weaver MAOM, HTL (ASCP) QIHC Date: Mon, 20 Apr 2015 19:06:17 +0200 From: j.benavi...@eae.csic.es To: histonet@lists.utsouthwestern.edu Subject: Re: FW: [Histonet] prevent wrinkles when cutting Hi there, I´m curious about the soaking thing. We have never done it in our lab. Which is the purpose to do it? Than, after facing the blocks, we chill them in a cold plate so, if wanting to do the soaking , when should we? I guess before placing them on the cold plate, but that may cause a bit of ice formation? Thanks a lot for your help Julio On 20/04/2015 18:40, Grantham, Andrea L - (algranth) wrote: Rachel, First off, are you chilling and soaking the blocks after you face them? Do that and see if there is a difference. Don't try to get many sections to your ribbons. Shoot for a smaller ribbon (5-6) sections that are good. Cut slowly but consistently. What microtome are you using? Are you using disposable blades and are they sharp? Don't expect them to cut well if you use the same blade to face the blocks. If you aren't using disposables, get some! They will make your life easier. You might try to find a histotech at a local hospital lab who might be able to give you a hands-on lesson. Don't despair! We all sat down at our microtomes those first times and suffered trying to get perfect sections. It takes practice. You might make some blank blocks or blocks with tissue you can spare to practice your cutting techniques. I used to do this with my students and it really helped them. Good luck! Andi G. From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Rachel M Gonzalez [rac...@gbi-inc.com] Sent: Monday, April 20, 2015 9:07 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] prevent wrinkles when cutting Hi Thursday was the first time I ever used a microtome I move to a lab that does not have someone dedicated to cutting. I already miss her. I have no problems getting ribbons of 10-30 sections long but the pieces are half the size of the original block. I am guessing they are wrinkling. What am I doing wrong? Thanks Rachel Senior Scientist ___ 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 ___ 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] prevent wrinkles when cutting
No ice forms in fixed, paraffin embedded tissue blocks at usual temperatures and length of chilling time and temperatures. Joelle Weaver MAOM, HTL (ASCP) QIHC Date: Mon, 20 Apr 2015 19:06:17 +0200 From: j.benavi...@eae.csic.es To: histonet@lists.utsouthwestern.edu Subject: Re: FW: [Histonet] prevent wrinkles when cutting Hi there, I´m curious about the soaking thing. We have never done it in our lab. Which is the purpose to do it? Than, after facing the blocks, we chill them in a cold plate so, if wanting to do the soaking , when should we? I guess before placing them on the cold plate, but that may cause a bit of ice formation? Thanks a lot for your help Julio On 20/04/2015 18:40, Grantham, Andrea L - (algranth) wrote: Rachel, First off, are you chilling and soaking the blocks after you face them? Do that and see if there is a difference. Don't try to get many sections to your ribbons. Shoot for a smaller ribbon (5-6) sections that are good. Cut slowly but consistently. What microtome are you using? Are you using disposable blades and are they sharp? Don't expect them to cut well if you use the same blade to face the blocks. If you aren't using disposables, get some! They will make your life easier. You might try to find a histotech at a local hospital lab who might be able to give you a hands-on lesson. Don't despair! We all sat down at our microtomes those first times and suffered trying to get perfect sections. It takes practice. You might make some blank blocks or blocks with tissue you can spare to practice your cutting techniques. I used to do this with my students and it really helped them. Good luck! Andi G. From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Rachel M Gonzalez [rac...@gbi-inc.com] Sent: Monday, April 20, 2015 9:07 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] prevent wrinkles when cutting Hi Thursday was the first time I ever used a microtome I move to a lab that does not have someone dedicated to cutting. I already miss her. I have no problems getting ribbons of 10-30 sections long but the pieces are half the size of the original block. I am guessing they are wrinkling. What am I doing wrong? Thanks Rachel Senior Scientist ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
