[Histonet] Hydroxyprobe-1 or Hydroxyprobe Omni kits for detectinghypoxia

[Jacqui Detmar]

Hi Gail.  We have used hypoxyprobe on mouse placentae and have seen
increased staining with one of our knockout mouse lines having defective
placental vasculature (compared with wildtype placentae).  We used the
rabbit polyclonal kit and it worked nicely, so your researcher should
not have a problem.  

Additionally, I have started some Hif1-alpha IHCs on mouse placental
sections and I am getting a similar staining pattern to that seen with
hypoxyprobe, so there does seem to be some biological validity to the
technique (although I need to keep tweaking this antibody).  This is an
important fact to consider b/c I know when my labmate presented this
work, she was harassed by the audience regarding the validity of the
results.  I'm sure it'll be no different upon manuscript submission and
review.  

Anyway, while your researcher may need to provide a qualifier for the
first set of experiments, subsequent experiments should not require this
(if results are repeatable) and the hypoxyprobe technique is *so* much
easier....I recommend it.

Jacqui

Jacqui Detmar, Post-doctoral Fellow
Samuel Lunenfeld Research Institute, room 876
Mount Sinai Hospital
600 University Avenue
Toronto, ON, Canada
M5G 1X5

phone:   416-586-4800 x2451/x2290
fax:        416-586-8588
email:     detmar <@t> mshri.on.ca

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gayle
Callis
Sent: Friday, May 30, 2008 1:29 PM
To: Histonet
Subject: [Histonet] Hydroxyprobe-1 or Hydroxyprobe Omni kits for
detectinghypoxia

Is anyone doing the staining for hypoxia using these kits
(Hydroxyprobe-1 or Hydroxyprobe Omni)?   I have a researcher (his
laboratory personnel is not experienced with immunostaining, and not
sure he has done it himself) wanting set up immunostaining on murine
lung showing hypoxia using one of these kits.  One avoids mouse on mouse
staining issues (Omni contains polyclonal rabbit primary) while the
other kit has success with using the mouse monoclonal primary
(Hydroxyprobe-1).   For a lab starting out with immunhistochemical
staining, avoiding the mouse on mouse issue may be a good introduction
to IHC, but maybe the Omni Rabbit polyclonal kit is not as ideal.  They
can certainly learn the ropes here.   

Questions:  

1.  Which kit do you prefer? 
2.   Fluorescence or chromogenic method?  With which fixation NBF or
frozen sections fixed with acetone? 
3.   In their website, they cite a protocol by Raleigh et al, where they
routinely use CSA (DAKO) for mouse antibodies for clinical samples.  Is
anyone using CSA as they did?  We would like to avoid this if possible,
and even use frozen section for enzyme IHC chromogenic methods. 
4.  Since mouse lung is the target tissue, we would like to use alkaline
phosphatase instead of HRP enzyme method?  Any comments?  Our lung
experts prefer alk phos over HRP due to high background issues with
peroxidase methods in the past.   

Any comments are welcome 

Gayle M. Callis
HTL/HT/MT(ASCP)
Bozeman MT 

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