Do  I understand right, that she was until now not able to prove, if the antibody works (no successful IHC) because of falling off the tissue?

How long do you dry the sections before IHC? temp?

adhesive slides? thickness of sections?

What method of HIER do you use? steamer, pressure cooker? waterbath? duration? temp?

It may just be a shot in the dark, but one month in EDTA-solution can be long enough for a removement of formalin-bindings. - so like a very long, gentle antigen-retrieval.

Have you tried to do the IHC without any retrieval at all?

Was the formalin freshly made? Or an old batch? If it was old a couple of weeks and was not buffered, it is possible that the pH was lowered by developing formic acid. That would lead to acid formalin, which may have a bad influence on the antigen.

Hi Gurdun,

She has done multiple versions of heat antigen retrieval ( none of which worked, since the heat made the tissue slide off, she put it on charged slides), so moved on to the proteolytic antigen retrieval, but the problem is that NO  company that supplies the antibody with RAT re activity has performed proteolytic antigen retrieval with this antibody. 

My biggest concern is that she fixed the tissue in formalin (10% non-buffered for 5 days , as this is the protocol lab usually uses for HISTOLOGY).  so she just assumed that this would be fine for immunohistochemistry too, I believe that there is permanent formalin  crosslinkage on proteins now , and heat retrieval does not work ( not sure it would even if heat did not effect the tissue).  

She did these before I came into the lab, and not many have experience with IHC here, so she followed protocols for regular histology staining performed here.

Any suggestions if anything at all can be done at this point?

Help and input much appreciated

Hi Yarina,

I suggest to test if the whole antigenicity of the samples is preserved, just to understand if there is a general problem of tissue processing that impairs antibody-antigen linking or if the problem is limited to RAGE antigenicity. You can try some normal bone markers (vimentin, osteonectin...) and some osteoclast markers (OSCAR, RANK...).

 5 days of formalin fixation is not a big deal, although because it was unbuffered, there might be acid hematin formation, causing black granular pigment in vessels. I'd be more worried about the one-month decal. But in any case your biggest problem is the slides coming off. Does she incubate the slide at 37 C overnight after cutting, before doing anything else ? If you can't get sections to stick, just try proteinase retrieval, it doesn't matter that companies haven't tried it with their antibody. Just try it without any retrieval too, see what you get. IHC parameters have to be developed and optimized in the lab anyway, the information that companies can provide about protocols for their antibodies is always just a general guide, and often not a very good one.

I copy/paste from histonet FYI.

-----Original Message-----

From: Danielle Crippen [mailto:dcrippen buckinstitute.org]

Sent: Wednesday, March 07, 2007 2:47 PM

To: Kang, Joseph J; histonet lists.utsouthwestern.edu

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

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

Hey guys, so just wanted to update on the protocol for this in case anyone runs into the same issues

We were able to successfully resolve the problem

The antigen retrieval was done at 65C overnight ( 12 h) in a citrate buffer and then followed with the appropriate quenching of peroxidase activity, blocking, primary and secondary antibody incubations and Vectastain ABC developer kit

While the leg of the bone ( Rat Tibia) was falling off slightly, the region of interest ( the growth plate of tibias head remained intact)

Thanks