Hi Lydia,
I think Tony Henwood has it exactly right in talking of DAB intensification.
The article he sites and several others show how much more sensitive DAB
intensification can make an ordinary iron reaction. And you are not looking in
bone marrow or spleen but somewhere where there is so little iron.
More than once I've been burned by research colleagues who gave me tissue
saying "x" should be upregulated and spend weeks looking for it until they say
"oop's, sorry-my bad". Intra-dermal injections can become sub-dermal. IP
injections can be sub-optimal with rough handling of mice. IV tail vein
injections can be missed. And MPTP is toxic and dangerous enough to work with
that I'd be fumbling around and nervous myself. If you are confident the model
is working by some secondary marker such as tyrosine hydroxylase
immunoreactivity then you are still are looking for minute quantities of iron.
Several easily available papers on that very mouse model tell you the limited
cell population to look for (one says-NOT in the big cells), and in a very
specific region using coronal sections (I always used a mouse brain mold to
section to be sure of the anatomical location) and several of the papers use
classical iron histochemistry followed by DAB intensification and their
procedures are in material and methods.
Reaction might be working after all-just have to focus in on very limited
reaction product. Good luck hunting.
Ray
Ray Koelling
PhenoPath Labs
Seattle, WA
----- Original Message -----
From: "Lydia Gunawan" <lydia.guna...@unimelb.edu.au>
To: Histonet@lists.utsouthwestern.edu
Sent: Tuesday, November 15, 2011 2:00:32 PM
Subject: [Histonet] help
Hi there, I am having trouble with Turnbull staining. Anybody can help me?
As I want to detect and quantify iron in the brain for Parkinson experiment, I
am using mice(C57Bl6) brain that were treated with MPTP injection. So, I have
been trying to stain those brain using paraffin section with Turnbull blue but
I have no luck.
FYI, I have been using 7% of Potassium ferricyanide in 3% HCL and I incubated
for 2 hours, 37'. I also did incubation in triton-x and H2O2 too but still not
getting any iron on my sections. Could anyone help me to solve my problem?
Thanks
Lydia Gunawan
Oxidation Biology Laboratory
Mental Health Research Institute
Melbourne Brain Centre
Corner Royal Pde and Genetics Lane
University of Melbourne, Level 4
Parkville, Vic 3010
email: lydia.guna...@unimelb.edu.au
_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
