Jane,

I have done a fair amount of immuno on mouse knees, they cut very well on a
cryostat with a sharp steel C-profile knife.  I was cutting STR/ORT mice (a
strain that develop spontaneous OA) and we sectioned them in the sagittal
plane.  It was important to freeze them with OCT in isopentane precooled
with nitrogen.  The snap freezing gets them real cold real quick, the OCT
supports the tissue during cutting.  The difficulty comes with keeping the
sections on the slide, mineralised bone does not stick well.  We used home
made APES coated slides, the gold standard these days is to use the
instrumedics tape transfer system (if you can afford it!).  Keep your immuno
as short as possible (use a 2 step indirect method if possible), and treat
the slides gently (I washed slides by immersion in buffer, not by squirting
the stuff on).  add some surfactant to your H2O2 block to remove surface
tension, O2 bubbles will be small and not lift your tissue from the slide.
We never bothered with decal of the bone, until after it was sectioned, and
then only for certain antigens.

You should get a whole bunch of replies on this topic, there are some really
good boneheads out there!

Regards,

Simon

Simon Smith B.Sc. AIBMS
Supervisor, Laboratory Resources
Skeletech, Inc.
22002 26th Ave SE, Suite 104
Bothell   WA   98021
Voice: (425) 424 2663   Fax:  (425) 424 2600
E-mail: ssmith@skeletech.com  


-----Original Message-----
From: Jane Chambers [mailto:jane.chambers@Agouron.COM]
Sent: Tuesday, May 23, 2000 5:53 PM
To: HistoNet Server
Subject: IHC on bone


Dear Histonet,

I have experience doing IHC on paraffin embedded tissues but not on
bone.  We are going to want to do IHC on knee joints of mice.  Some of
the antibodies will work on paraffin embedded tissue; others on frozen
tissue.  We will contract this work out and I will get specifics from
whomever we go with.  However, I wanted to get some general information
just so I have an idea of what to plan for.  Do you just snap freeze the
bone in liquid nitrogen or place it some sort of media and then in a -80
freezer?  When cutting for frozen sections, do you have to decalcify the
bone first?  What type of microtome do you use?  Do most labs that do
IHC do it on frozen bone?

Thank you,

Jane Chambers