[Histonet] help

[koellingr <@t> comcast.net]

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.gunawan <@t> unimelb.edu.au> 
To: Histonet <@t> 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.gunawan <@t> unimelb.edu.au 


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