[Histonet] Paraformaldehyde Fixed Tissue

[Michael Gudo]

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 at 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.Maluenda at baker.edu.au W www.baker.edu.au
> 
> -----Original Message-----
> From: Sandra Cheasty [mailto:sandra.cheasty at wisc.edu]
> Sent: Tuesday, 29 August 2017 1:30 AM
> To: Histonet (histonet at lists.utsouthwestern.edu) <histonet at 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
> 
> 
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