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 <[email protected]> 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
> [email protected]
> www.premierlab.com
>
> Ship to Address:
>
> Premier Laboratory, LLC
> 1567 Skyway Drive, Unit E
> Longmont, CO 80504
>
> -----Original Message-----
> From: [email protected]
> [mailto:[email protected]]
> <[email protected]]> On Behalf Of dusko trajkovic
> Sent: Monday, January 20, 2014 12:47 PM
> To: Esther C Peters; [email protected];
> [email protected]
> 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 <[email protected]>
> To: "[email protected]"
> <[email protected]>; "[email protected]"
> <[email protected]>; dusko trajkovic <[email protected]>
> 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: [email protected]
> <[email protected]> on behalf of dusko trajkovic
> <[email protected]>
> Sent: Monday, January 20, 2014 1:58 PM
> To: [email protected]; [email protected]
> 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: "[email protected]"
> <[email protected]>
> To: [email protected]
> 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
>
> _______________________________________________
> Histonet mailing list
> [email protected]
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