[Histonet] IHC bone tissue adherence issues

[Danielle Crippen]

Hi Joe,

We have had great success doing the following with mouse bones...

Leave bone samples immersed in PFA for at ~7 days...then proceed

DECALCIFICATION

-	Decalcify in Immunocal (Decal Chem), with agitation, changing solution and checking for endpoint daily.
o	To Check for endpoint mix 4 parts spent decal solution (formic acid) to 1 part 5% ammonium oxalate (w/v)...if there's a white calcium oxalate ppt...another day of decalcification is needed
o	Once decalcification is complete, run bones under running tap water for a few minutes, then wash in tap water for 1-4 hours
o	Bones can be stored in PBS until processing can begin

PROCESSING

-	70% EtOH 1.5 hours
-	80% EtOH 1.5 hours
-	95% EtOH 2x 1.5 hours
-	100% EtOH 2x 1.5 hours
-	Clearite 3 2x1.5 hours
-	Tissue Prep 2 paraffin 4x1.5 hours 60 degrees under vacuum
-	Embed in Tissue Prep 2

Then we section 7um sections onto plus slides (we use Starfrost plus from Mercedes Medical cat# MER7255-90-WH).  Let them airdry o/n at RT.  Next day bake them lying flat at 60degrees C for 1 hour before IHC.

We use a standard IHC protocol only we replace our antigen retrieval protocol with Decal Retrieval soln (Biogenex HK089-5K) which doesn't require heating.

Our technique doesn't seem greatly different from anything you've tried...but our bone tissues always stay in tact throughout staining...we've never had a problem...

We've only used mouse femur and tibia..so there may be a difference b/t our samples..different bone, different animal???

Hope this is helpful...

d

Danielle Crippen
Morphology and Imaging Core Manager
Buck Institute for Age Research
8001 Redwood Blvd.
Novato, CA 94945
415-209-2046
dcrippen <@t> buckinstitute.org



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Kang,
Joseph J
Sent: Wednesday, March 07, 2007 11:09 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC bone tissue adherence issues


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




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