Re: [Histonet] Paraformaldehyde Fixed Tissue
Dear Sandra, Dear Ana, in our lab for tissues for which we have enough time for infiltration we fix for at least 48 hours or more in NBF or PFA (both with 4 % reactive formaldehyde, which is equivalent to 10% „neutral buffered formaline“) and then first wash in tap water for a minimum of 1-2 hours, then to 30%, 50%, 60%, 70%, 80%, 90% 96% (2 times), 2-Propanol (2-times) and then 2-3 time xylene, before embedding in paraffine. The times are just a few hours per step or shorter, and we have quite good results especially for brain and spinal chord. Best regards Michael > Am 29.08.2017 um 02:06 schrieb Ana Maluenda via Histonet > <histonet@lists.utsouthwestern.edu>: > > Hi Sandra, > > I haven't myself particularly worked with brain and spinal cord, but majority > of my protocols in my old job used fixation in 4% PFA (24 hours at 4-8oC) and > routinely process (or transfer to graded EtOH, if not processing immediately). > However, our routine process didn't include a first step in 10% NBF, since > PFA plays the role of fixation. Therefore, after PFA, we would have 70% EtOH, > 80% EtOH, 95% ETOH, 2x EtOH the Xylenes and wax [assuming you are referring > to FFPE?]. > Also mouse tissue can be small and delicate, so I remember running liver, > spleen, kidney and thymus (soft tissues I worked with) in a short cycle > (similar to what I would do for biopsies). > > Hope this helps! > > Kind regards, > > Ana > > > Ana Maluenda > Research Assistant > Atherothrombosis and Vascular Biology Laboratory > > Baker Heart and Diabetes Institute > 75 Commercial Road, Melbourne VIC 3004 > P (03) 8532 1359 E ana.malue...@baker.edu.au W www.baker.edu.au > > -Original Message- > From: Sandra Cheasty [mailto:sandra.chea...@wisc.edu] > Sent: Tuesday, 29 August 2017 1:30 AM > To: Histonet (histonet@lists.utsouthwestern.edu) > <histonet@lists.utsouthwestern.edu> > Subject: [Histonet] Paraformaldehyde Fixed Tissue > > Hi all, > We are having difficulty sectioning mouse tissue, (brain, > spinal cord, liver, and spleen), on paraformaldehyde fixed tissue. Has anyone > had issues with paraformaldehyde fixed tissue? They were processed routinely, > starting in 10% NBF, with other tissues, and we are cutting them at 3u. > Thank you! > Sandy > > Sandra J. Cheasty, HT (ASCP) > Histology & Necropsy Supervisor > UW-Madison, School of Veterinary Medicine > > > Protecting your privacy is important to us. The Baker Heart and Diabetes > Institute will handle your information in accordance with the Privacy Act > 1988 (Cth) and its Privacy Policy which is available at www.baker.edu.au or > on request by contacting priv...@baker.edu.au or by calling 1800 838 498. The > Privacy Policy also explains how you can access and correct your personal > information, or make a complaint about a breach of the Australian Privacy > Principles. bidipp2014.0.1a > -- > Message protected by MailGuard: e-mail anti-virus, anti-spam and content > filtering.http://www.mailguard.com.au/mg > > > ___ > 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] Paraformaldehyde Fixed Tissue
Hi Sandra, I haven't myself particularly worked with brain and spinal cord, but majority of my protocols in my old job used fixation in 4% PFA (24 hours at 4-8oC) and routinely process (or transfer to graded EtOH, if not processing immediately). However, our routine process didn't include a first step in 10% NBF, since PFA plays the role of fixation. Therefore, after PFA, we would have 70% EtOH, 80% EtOH, 95% ETOH, 2x EtOH the Xylenes and wax [assuming you are referring to FFPE?]. Also mouse tissue can be small and delicate, so I remember running liver, spleen, kidney and thymus (soft tissues I worked with) in a short cycle (similar to what I would do for biopsies). Hope this helps! Kind regards, Ana Ana Maluenda Research Assistant Atherothrombosis and Vascular Biology Laboratory Baker Heart and Diabetes Institute 75 Commercial Road, Melbourne VIC 3004 P (03) 8532 1359 E ana.malue...@baker.edu.au W www.baker.edu.au -Original Message- From: Sandra Cheasty [mailto:sandra.chea...@wisc.edu] Sent: Tuesday, 29 August 2017 1:30 AM To: Histonet (histonet@lists.utsouthwestern.edu) <histonet@lists.utsouthwestern.edu> Subject: [Histonet] Paraformaldehyde Fixed Tissue Hi all, We are having difficulty sectioning mouse tissue, (brain, spinal cord, liver, and spleen), on paraformaldehyde fixed tissue. Has anyone had issues with paraformaldehyde fixed tissue? They were processed routinely, starting in 10% NBF, with other tissues, and we are cutting them at 3u. Thank you! Sandy Sandra J. Cheasty, HT (ASCP) Histology & Necropsy Supervisor UW-Madison, School of Veterinary Medicine Protecting your privacy is important to us. The Baker Heart and Diabetes Institute will handle your information in accordance with the Privacy Act 1988 (Cth) and its Privacy Policy which is available at www.baker.edu.au or on request by contacting priv...@baker.edu.au or by calling 1800 838 498. The Privacy Policy also explains how you can access and correct your personal information, or make a complaint about a breach of the Australian Privacy Principles. bidipp2014.0.1a -- Message protected by MailGuard: e-mail anti-virus, anti-spam and content filtering.http://www.mailguard.com.au/mg ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Paraformaldehyde Fixed Tissue
Hi Sandra, What are the times for each processing station? Animal tissue is much leaner, so it's easy to over process and become brittle to section. Best, Tina Van Meter On Mon, Aug 28, 2017 at 11:30 AM, Sandra Cheasty via Histonet < histonet@lists.utsouthwestern.edu> wrote: > Hi all, > We are having difficulty sectioning mouse tissue, (brain, > spinal cord, liver, and spleen), on paraformaldehyde fixed tissue. Has > anyone had issues with paraformaldehyde fixed tissue? They were processed > routinely, starting in 10% NBF, with other tissues, and we are cutting them > at 3u. > Thank you! > Sandy > > Sandra J. Cheasty, HT (ASCP) > Histology & Necropsy Supervisor > UW-Madison, School of Veterinary Medicine > > ___ > 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] Paraformaldehyde Fixed Tissue
Hi all, We are having difficulty sectioning mouse tissue, (brain, spinal cord, liver, and spleen), on paraformaldehyde fixed tissue. Has anyone had issues with paraformaldehyde fixed tissue? They were processed routinely, starting in 10% NBF, with other tissues, and we are cutting them at 3u. Thank you! Sandy Sandra J. Cheasty, HT (ASCP) Histology & Necropsy Supervisor UW-Madison, School of Veterinary Medicine ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
