[Histonet] Quick frozen sectioning and IMHC questions

Tue Aug 24 17:57:56 CDT 2010

Patrick,

Bring sections out of freezer and air dry at RT immediately, preferably lay
on a countertop to warm as quickly as possible.  They may look a little
"frosty" but let them warm up for a few minutes - what you see is some water
condensation.  You cannot wash these sections in buffer before fixation, so
mark with PAP pen then fix the air dried sections.  After that, go to buffer
and then stain.  IF you wash before fixation, you will lose an unfixed
frozen  section off the slide.  

We prefer to air dry unfixed fresh tissue frozen sections (I presume that is
what you did here?) for a minimum of 30 minutes, then put into a storage box
that holds ONE days staining run.  We put a little bag of 16 mesh silica gel
in the box with sections. Take box from freezer, and let the sections come
to room temperature for 20 minutes or longer.  Open box, mark with PAP pen,
fix and stain.  If you open the box immediately after removing from freezer,
you will get water condensation.  And if you open a box, remove some
sections, then return box to freezer, you get a thaw/freeze of sections.
Water condensation can damage sections and thaw/freeze will eventually
damage antigens - I would avoid both.  If storing fresh tissue frozen
sections as positive and negative controls, use the 5 slide mailers with
lids, and take out a mailer to warm up to RT.  This keeps other sections
frozen until use.  We put the mailers w/ slides in a zip lock baggie for
quick removal of a few sections to avoid opening and closing a large box.  

As for BSA, we no longer use it for any immunostaining
procedures/buffers/diluents,blocking reagents, etc but if you must use it,
there is Jackson's immunoglobulin/protease free.  This is also available
from Sigma, high quality for molecular biology procedures and in larger
quantity. We use only Tween 20 in our buffers now. There is a publication
(which I would be happy to send in pdf) about the effects of BSA with
primary and secondary antibodies in AIMM journal and why it causes IHC
problems. We prefer a normal serum block matched to host of secondary
antibody. 

Once sections are at RT, they stay at RT unless fixed in cold acetone (4C
for 10 min, then air dry again at RT) then all staining, etc is done at RT. 

We never store a frozen section in a cryostat, it goes from pick up to RT
for air drying then freezer storage (-80C, -70C is good). 

Have fun on your new job.  

Gayle Callis
HTL/HT/MT(ASCP)
Bozeman MT     

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lewis,
Patrick
Sent: Tuesday, August 24, 2010 3:50 PM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Quick frozen sectioning and IMHC questions

Hi everyone,

I cut my first frozen sections, human tonsil, and I have stored them at
-70.  When I take them out to use, should I bring them to -20C and fix
them right away?  Or should I bring them to room temp and then pap
pen/wash/block/wash them then fix them before adding my primary
anti-body.

My original plan was/is to bring them to room temp, pap pen them and
then wash/block/wash fix.  I am asking for your advice as my boss is
away for two weeks and the protocol he left me with is a bit vague and
requires some modifications to fit the research that we are doing.

I was just wondering if there is a compelling reason to keep the
sections at/near -20 after sectioning up through the fixation before
starting the Immunohistochemistry.

Also, we are using BSA as one of the components in our blocking solution
as well as a component in our wash solution.  Is there a danger in using
lower/higher quality BSA?   I'm a little worried that our BSA might
contribute to increased background if its quality is not high enough.

The BSA we may use is A-8327 Sigma, and I am thinking this might be
better A3294, or one of these (A9647,A7906,A6793)

This is a new job for me, and its been a long while since I have done
any Immunohistochemistries.so I am going over the reagents we have on
hand while I refresh my memory on immunohistochemistry and sectioning
techniques.

Thanks for all your support

Patrick

Patrick Lewis

Research Associate II-Bench| Infections and Prematurity

Seattle Children's Research Institute

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