[Histonet] help

John Kiernan jkiernan <@t> uwo.ca
Wed Nov 16 13:48:22 CST 2011


The Turnbull's blue method as you describe it will not detect iron in tissues. 
Potassium ferricyanide will give a blue precipitate only with iron(II) (ferrous). In tissues, the iron is present as iron(III) (ferric) in such proteins as ferritin and haemosiderin. The iron of haemoglobin, though abundant, cannot be released by acid to react as Fe(II) or Fe(III) ions. The easiest way to detect Fe(III) is with potassium ferrocyanide - 0.05M in 0.2M HCl, for 30m - which gives a Prussian blue deposit (Perls' reaction).  If you want to do a Turnbull's blue method, which some say is a bit more sensitive, you must first reduce all the Fe(III) in the tissue to Fe(II) with dilute ammonium sulphide. All the iron then ends up as precipitated FeS, which will react with an acidified potassium ferricyanide solution to produce Turnbull's blue.
In fact, Prussian and Turnbull's blues are the same compound (see inorganic chemistry textbooks). A faint or invisible reaction product can be amplified because this pigment behaves like peroxidase, catalyzing the oxidation of DAB by H2O2 (see e.g. Connor et al. 1995). The sensitivity can be further increased with chemical tricks to change the brown oxidation product of DAB into larger quantities of black stuff (Moos & Mollgard 1993).
Connor JR et 4 al (1995) A histochemical study of iron-positive cells in the developing rat brain. J. Comp. Neurol. 355:111-123. 
Moos T & Mollgard K (1993) A sensitive post-DAB enhancement technique for demonstration of iron in the central nervous system. Histochemistry 99:471-475. 
John Kiernan 
Anatomy, UWO 
London, Canada 
= = =

----- 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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