[Histonet] Histogel

Wed Jan 22 08:28:42 CST 2014

Thank you, John!

For some strange reason, the URL in your message got garbled/multiplied, but I just took this part:

http://www.thermo.com/eThermo/CMA/PDFs/Various/File_9759.pdf

and that worked.

The enrobing I do is to trap debris on the surface of a coral skeleton at the interface between "apparently healthy" tissue and "bare" skeleton in studying the tissue loss diseases of the corals. After solidifying the agarose, I decal the sample in neutral pH 10% EDTA, then cut ("breadloaf-style") the agarose-enrobed tissue block into ~2-mm slices and put each in a cassette for processing. Perhaps my problem is the processing time needs to be increased to be sure to remove all the water from the agarose and infiltrate it completely.

I don't know what the instructions mean by "non-porous filter paper," either, has anyone figured this out?

Esther
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu <histonet-bounces <@t> lists.utsouthwestern.edu> on behalf of John Kiernan <jkiernan <@t> uwo.ca>
Sent: Tuesday, January 21, 2014 8:54 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Histogel

The document at http://lists.utsouthwestern.edu/mailman/listinfo/histonet(http://www.thermo.com/eThermo/CMA/PDFs/Various/File_9759.pdf">http://www.thermo.com/eThermo/CMA/PDFs/Various/File_9759.pdf gives a thorough description of Histogel, and even says what it is - hydroxyethyl agarose.  In the detailed instructions for various uses, the only confusing thing is the requirement for "non-porous filter paper"!

John Kiernan
Anatomy, UWO
London, Canada
= = =
On 20/01/14, Elizabeth Chlipala <liz <@t> premierlab.com> wrote:
>  Esther
>
> I agree with Dusko, I fix before I put in histogel and again after the sample is placed in histogel, we have no formalin on our tissue processor, we start in 50% alcohol.  I also process on a longer processing cycle, 1 hour per station and similar to Dusko's - denatured ethanol, xylene and paraplast and paraplast extra to embed.  I've never had a problem (such as overprocessed tissue) with the histogel or the sample embedded in the histogel with the longer processing cycle.  Most of the samples we process are cell blocks or tissue fragments such as micronized tissue constructs, which are like powder when we receive them.
>
> 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
> liz <@t> premierlab.com
> www.premierlab.com
>
> Ship to Address:
>
> Premier Laboratory, LLC
> 1567 Skyway Drive, Unit E
> Longmont, CO 80504
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] <histonet-bounces <@t> lists.utsouthwestern.edu]> On Behalf Of dusko trajkovic
> Sent: Monday, January 20, 2014 12:47 PM
> To: Esther C Peters; Jennifer.Arcand-Johnson <@t> genzyme.com; histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] Histogel
>
> Esther,
> I mainly process cells, which have been spun down into a small pellet. Also mouse DRG's and other very small tissues. I would consider this delicate, so do not be afraid to use a longer processing program. Histogel/Agurose is what needs longer dehydrating steps.
> We do not use any substitute reagents, so in that aspect I cannot tell you how they will affect the processing. Our lab uses ethanol, xylene, and Paraplast paraffin. Try a test run and let me know if you were able to get successful results.
> Have a good Monday!
> Dusko
>
>
> ________________________________
>  From: Esther C Peters <epeters2 <@t> gmu.edu>
> To: "Jennifer.Arcand-Johnson <@t> genzyme.com" <Jennifer.Arcand-Johnson <@t> genzyme.com>; "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>; dusko trajkovic <dunatrsd <@t> sbcglobal.net>
> Sent: Monday, January 20, 2014 11:15 AM
> Subject: RE: [Histonet] Histogel
>
>
> Thank you, Dusko!
>
> I have had the same problem with 1.5% agarose, and I tried starting the dehydration with 30% to 50% to 70% ethanol, and using different xylene substitutes. It appears that the variable whitening and shrinking happens after 100% reagent alcohol and in the xylene substitute (now using Richard-Allan Clear-Rite3). I've wondered if slow infiltration was the issue.  I guess we'll try this longer processing, but I also work with delicate tissues that normally would be a short run (15 min in each reagent). Are your tissues thin/delicate/biopsy or cell preps or organ samples? No effect on them?
>
> Esther
>
> Esther C. Peters, Ph.D.
> Assistant Professor
> Environmental Science & Policy
> George Mason University
> 4400 University Drive, MS 5F2
> Fairfax, VA 22030-4444
> ________________________________________
> From: histonet-bounces <@t> lists.utsouthwestern.edu <histonet-bounces <@t> lists.utsouthwestern.edu> on behalf of dusko trajkovic <dunatrsd <@t> sbcglobal.net>
> Sent: Monday, January 20, 2014 1:58 PM
> To: Jennifer.Arcand-Johnson <@t> genzyme.com; histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] Histogel
>
> Jennifer,
> You might have seen one of my posts from 2-3 years ago. I had the exact same problems you described. Could not get anyone to come up with a solution. I ran various programs on our VIP and finally came up with a solution.
> Fix your specimens as you normally would do. Drain of the fixative add your histogel (dissolved in hot water, which you have been doing), fill the mold with the histogel. Let solidify on ice or 4C in fridge. Place the solid histogel in a cassette and process on a 12 hour program. Since I have instituted this procedure, have not had one bad block to date. Longer processing is the answer, and nothing else.
> Good Luck.
> Dusko Trajkovic
> Pfizer Inc. La Jolla
> 858-638-6202
>
>
> ________________________________
> From: "Jennifer.Arcand-Johnson <@t> genzyme.com" <Jennifer.Arcand-Johnson <@t> genzyme.com>
> To: histonet <@t> lists.utsouthwestern.edu
> Sent: Monday, January 20, 2014 7:56 AM
> Subject: [Histonet] Histogel
>
>
> Dear Histonetters,
>
> I have been reading up on the archives for info on Histogel.  Previous posts discuss how they had problems with it - some samples would come out great and some would shrivel up or even dissolve.
> These posts on the Histogel were from a few years ago and was hoping, and praying that someone out there may have solved this issue and have a little info you could share with me on this subject.  Did anyone out there ever figure out how to get consistent results?
> I have spoken with RA Scientific and they have no additional insights.
>
> Here is the background:  I have used Histogel for about 4 years now.  In September of last year, we started seeing the shriveling Histogel samples.  Like others who posted, it was random.  I could embed two serial pieces of nerve, from the same mouse, into two blocks and one would shrivel and one would look great.  So I have tried many things, always in multiples of 3 or more per condition per run...
>
> Fixing in formalin only, embedding in Histogel and storing in PBS until processing
> Fixing in formalin only, embedding in Histogel and storing in 40% reagent alcohol until processing (the first step of our processor is 40%)
> Fixing in formalin only, embedding in Histogel and storing in formalin until processing
> Fixing in formalin, rinsing in 40% alcohol, embedding in Histogel, and storing in 40% or formalin until processing
> For all of these conditions, I have tried using a small cycle (30 min/bath) and a biopsy cycle (15 minutes/bath).
> Once processed, there was no rhyme or reason to the results.  Some blocks looked great; others within the same group looked shriveled.  Sometimes the blocks were white, sometimes they were clear.
>
> Next, I thought it was my pre-processing - so I heated the Histogel in a water bath, rather than microwaving.  That way all of the samples were embedded with the Histogel at the same temperature - about 55 degrees.
> Again, no rhyme or reason, some looked good, some looked bad.
>
> Lastly I thought that maybe I was carrying over too much liquid from my sample to the Histogel so I tried the following:
> I made Histogel only blocks or added 3-4 drops of 40% alcohol or formalin to the liquid Histogel before a tissue/cell free block was made.
> Yep, you guessed it - no luck.  Some looked good, some looked shriveled.
>
> So here I have this great tool to embed tiny samples, but I am afraid to use it because I don't know if it will work or shrivel!  Can anyone out there help me?
> Thanks,
> Jenn
>
>
> Jennifer Johnson
>
> Staff Scientist
>
> Genzyme, a Sanofi Company
>
> Department of Pathology
>
> 5 Mountain Road
>
> Framingham, MA 01701-9322
>
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