RE: [Histonet] mordanting in Bouin's, storage in 70%, processingmouse tissues
Thanks for the suggestions. Is there any particular reason for the low melting point paraffin?
> From: Nancy Lemke
> Sent: Wednesday, June 21, 2006 2:35 PM
> To: Wurdak, Elizabeth
> Subject: Re: [Histonet] mordanting in Bouin's, storage in 70%, processingmouse tissues
> Question 3:
> Depending on the thickness of your mouse sections, even if you are attempting whole organs, you are over processing everything. If you cut your organs into blocks of tissue no thicker than 2-3mm you can cut your time in half or less. Starting with 50% ethanol, 70%, 2x95%, 3x100%, 2xXylene and 4 x paraffin (low melting point of 54-56C, set paraffin baths to 57C), you can have all alcohols at 25 minutes, each xylene at 30 minutes and each paraffin at 20 minutes. The tissues will still be dry and brittle but no where near as dry as they must be with your protocol. Do not use heat on any solution, only on paraffin baths, but use pressure/vacuum on all stations if your processor allows. Try one mouse this way and then compare cutting to your old processing schedule. All of this assumes that your tissues are well fixed, either perfused followed by immersion for a total of no more than 24 hours for IHC or if immersed only,24-36 hours.
> Nancy Lemke
> Research Coordinator
> Hermelin Brain Tumor Center
> Henry Ford Hospital
> -----Original message-----
> From: "Wurdak, Elizabeth" EWURDAK@CSBSJU.EDU
> Date: Wed, 21 Jun 2006 15:23:11 -0400
> To: "Histonet (E-mail)" email@example.com
> Subject: [Histonet] mordanting in Bouin's, storage in 70%, processingmouse tissues
> > Hello,
> > I learned of this listserve group at the recent meeting of the > Biological Stain Commission. I wanted to join immediately because I > teach histology to undergraduate students. We work through the whole > paraffin technique in lab using manual methods. We are always coming up > with some questions. Here are a few:
> > 1. An attendee at the Stain Commission meeting mentioned using Bouin's > fixative as a mordant prior to Mallory staining of formalin fixed > tissues. We have been using saturated mercuric chloride in 5% acetic > acid. Is Bouin's better? Is it less toxic? For how long should the > sections be immersed in it? Do you post-treat with lithium carbonate to > remove the yellow picric acid before proceeding to the next step?
> > 2. How long can you store formalin fixed tissues in 70% alcohol? Is it > best to store them at room temperature or the refrigerator?
> > 3. Our mouse tissue blocks are coming out brittle, dry and hard to > section with the exception of lung and testis samples. I attributed > this to incomplete paraffin infiltration, but lengthening the parafffin > baths has not helped. It was suggested to me that perhaps we are > dehydrating too long. We have been using 3 changes of a 100% alcohol > for 1 hour each. What would you recommend?
> > Many thanks in advance for your suggestions.
> > Elizabeth
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