[Histonet] Agarose blocks

[Margaret Blount]

I have cut lots of agar blocks and used the normal tissue processing
schedules of whichever lab I worked in. I usually post fixed the agar block
in Neutral Buffered Formalin for a few hours to a day after embedding
depending upon how well fixed the sample was prior to embedding in the agar
and processing. I do think your client ought to have told you what was
embedded in the agarose else how do you know how much to trim or what the
orientation is? I have used this procedure to orientate isolated hair
follicles and flat intestine samples so that I could get sections of precise
areas, so it was essential to have all the available information about the
sample.

In difficult cases, I hand processed the agar blocks to paraffin, but this
should not be necessary if you have a process that is suited to the tissue
sample itself. I always used 2% w/v aqueous agar (Difco) and maintained it
at 55 to 60C in a waterbath to keep it molten. I made a mould suited to the
sample, e.g. a cut off syringe, pipetted a layer of agar into it and allowed
it to start gelling, I then layered on my sample so as to orientate it
correctly, then pipetted a few more drops of agar on top, taking care to
allow it to cool sufficiently to avoid "cooking" my sample. To achieve the
latter, I drew up some agar into a pastette and held it at room temperature
for a few seconds to allow it to cool, trial and error taught me how long to
do this. 

I have to say that I have used agar for both paraffin and frozen sectioning
successfully. My hand process went through 70% ethanol, 3 changes of
absolute ethanol and 1 of histoclear each step being 1 hour, then overnight
in histoclear, followed by a further histoclear of 1 hour, 2 changes of wax
in the wax oven for 1 hour then embed. This worked well for me. 

I hope this helps.

Margaret

Margaret Blount
Chief Technician
Clinical Biochemistry
University of Cambridge
Addenbrooke's Hospital
Hills Road
Cambridge
CB2 2QR 
-----Original Message-----
From: Roberta Horner [mailto:rjr6 <@t> psu.edu]
Sent: Wednesday, July 21, 2004 6:39 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Agarose blocks


I just received tissue in agarose and I tried cutting a couple of them with
very little luck.  I have never worked with this before.  Is there a trick
to sectioning it?  Should it have been processed differently?  I didn't know
what was in the block until I embedded it.

Thank you for any help.

Roberta Horner HT/HTL

 

Animal Diagnostic Lab

Penn State University

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