[Histonet] Histogel

[Jennifer.Arcand-Johnson <@t> genzyme.com]

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