[Histonet] IHC bone tissue adherence issues

[Gayle Callis]

Joseph,

This was discussed recently on Histonet so check out the Archives too. You 
did not say if you are using heat retrieval or enzyme digestion?  Some 
people prefer the gentler temperatures of digestion - I recall people using 
that instead of heat retrieval.

With Cryojane tape transfer, you can improve bone adherence to polymer 
slide by flashing the UV light twice, and I have done it three times.  Be 
careful to let the light source capacitor build up charge, so wait a bit 
between flashes.  Also, be sure your tungsten carbide d profile is 
absolutely the sharpest it can be in order to obtain very flat sections - 
it is important to have the section in total contact with the tape at 
initial rolling and a slide at transfer.  You can go from 1/2x to 1X, while 
4X polymer slides are super gooey but may be necessary to hold the section 
down.  We like the 1/2 X, but do have the others in case we run into major 
problems.

It may help to NOT dry the bone sections in a hot oven, but to lay sections 
on slides FLAT on a 37C - 40C slide warmer or on a slide tray in a 37C 
oven, overnight or longer.  Hot temperatures tend to dry out the bone 
excessively, in particular the cartilage.  Overdried bone sections often 
release (have had it happen too many times) even during routine 
staining.  Whenever possible, we dry at the lower temperature for several 
days.  It does test our patience.

Hope this helps

At 12:09 PM 3/7/2007, you wrote:
>Dear Histoneters,
>I have a great favor to ask.
>I need to find out what procedures I can use to keep bone sections
>intact on glass slides during IHC staining.
>There seems to be a lot of tissue lifting or loss during antigen
>retrieval. I am currently using plus slides on regular paraffin sections
>and leaving them in the oven for up to 24 hours with no success. I also
>attempted using the Instrumedics tape transfer system, which seems to
>retain more tissue than regular sections, but with similar results.
>We also attempted doing IHC on frozen knee joints using the tape
>transfer system to skip the antigen retrieval steps without success.
>Any positive feedback on the matter is greatly appreciated.
>
>Joe

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)