RE: [Histonet] IHC bone tissue adherence issues

From:"Kang, Joseph J"

Thanks for responding to my email.
Before we go further could you tell me what PFA is?
We typically do work on both rats and mice knee joints and paws.
We typically fix them in formalin for 24 hours for mouse and 48 hours
for rats, then we transfer in 70%ETOH for a couple of days before we
place them in Immunocal.
I will try your processing schedule for the mouse bones, but do you
happen to have a rat processing schedule by any chance?

In addition, we do a lot of method development for IHC, do you think
that the decal retrieval solution could be used for many IHC staining?

Thanks for your information.
I will try it out as well.


-----Original Message-----
From: Danielle Crippen [] 
Sent: Wednesday, March 07, 2007 2:47 PM
To: Kang, Joseph J;
Subject: RE: [Histonet] IHC bone tissue adherence issues

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


-	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


-	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 there may be a difference b/t
our samples..different bone, different animal???

Hope this is helpful...


Danielle Crippen
Morphology and Imaging Core Manager
Buck Institute for Age Research
8001 Redwood Blvd.
Novato, CA 94945

-----Original Message-----
[]On Behalf Of Kang,
Joseph J
Sent: Wednesday, March 07, 2007 11:09 AM
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.


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