[Histonet] IHC on N-cadherin, CD146, CD144,ELF97 TRAcP.

[Andrea T. Hooper]

Hi Julia,
 
I am sorry it's taken me months to reply to this email and hopefully it's not too late. I am only catching up on Histonet messages recently.
 
I stain for VE-cadherin, CD144 in murine bone routinely. R&D Systems goat anti-mouse VE-cadherin works very well in paraffin and frozen murine bone. 
 
If doing paraffin, I remove femurs/tibias from mice and fix in 4% PFA overnight at 4 deg C (can also fix 2-4h at RT). I decal in Richard Allan decal solution for 1h, sometimes 1.5h if femurs are especially large and remain hard after 1h. Wash well in dH20 and process into paraffin. I antigen retrieve in DAKO Target Retrieval Solution for 10 min at 95-99 degC in veggie steamer with 10 min cool down. 
 
Frozen sections for immunofluorescence are fixed in 4% PFA as above. Then decal in 10% EDTA for 5 days.  30% sucrose protect overnight at 4 deg C and snap freeze in OCT. 
 
It works well with both protocols.
 
For both frozens and paraffin, primary is at 2-4 ug/ml followed by very specific biotinylated secondary against goat IgG (Jackson Immunoresearch, minimally cross reactive to other species). Then use strep-HRP (Jackson IR) and DAB+ (DAKO) to develop.

Please let me know if you need more details.
 
Best,
Andrea Hooper
 

--- On Thu, 11/5/09, julia hough <julia.m.hough <@t> hotmail.co.uk> wrote:


From: julia hough <julia.m.hough <@t> hotmail.co.uk>
Subject: [Histonet] IHC on N-cadherin, CD146, CD144,ELF97 TRAcP.
To: histonet <@t> lists.utsouthwestern.edu
Date: Thursday, November 5, 2009, 3:38 PM





Dear histonetters.



I am trying to perform several new IHC stains on mouse
tibia. However, I am having trouble finding literature on some of them. I would
like antibodies that work on mouse tissue/bone that are preferably conjugated
to a fluorchrome. 



If anyone has any potentially protocols or information where
I could get primary antibodies unconjugated/conjugated for the following that
would be lovely:



N-cadherin, CD146, CD144,ELF97 TRAcP.



Thanks for your time.



Julia

                          
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