[Histonet] RE: Nitroblue Tetrazolium Chloride

[Houston, Ronald]

Nitro BT is a tetrazolium salt used in many dehydrogenase enzyme histochemical techniques. It is used in powder form (I would recommend the Nitrotetrazolium Blue chloride from Sigma, cat N6876).



The Nitro BT test is used for the demonstration of early myocardial infarction in gross hearts at necropsy by a dehydrogenase enzyme histochemical reaction on slices on unfixed heart.

In brief, a redox indicator is reduced by mitochondrial enzymes in cardiac muscle cells producing a blue staining reaction in all but infarcted areas. The loss of enzyme activity from the tissue defines the damaged heart muscle



Incubation Medium:



      Nitro BT                            50mg

      0.2M Tris-HCl buffer, pH 7.4        100ml

      Sodium cyanide                      50mg

      β-NAD                               100mg

   Adjust pH to 7.1

The stock solution can be kept for several weeks at 4C



Protocol:

1.                Rinse slices of myocardium in cold running water to remove the blood.

2.                Incubate slices at 37C for 30 minutes

3.                Wash in running water

4.                Store in formol saline


Result:
      Normal myocardium       - dark blue
Areas of infarcted muscle show diminished staining, the degree of which depends on age of infarct

Notes:
Loss of staining can be detected as early as 1-5 hr after clinically determined onset of infarct. Material can be tested for 3 days after death and even longer if stored at 4C

Cyanide is added to the incubation medium to bind keto acids, produced during the dehydrogenation of some hydroxy acids, which may inhibit the dehydrogenase reaction.

NAD is added to the reaction medium as it is lost from the tissue rapidly post-mortem, and greatly improves the sensitivity of the reaction.

Some reaction medium call for the addition of the electron transfer mediator phenazine methosulfate (PMS), ostensibly to increase the reaction velocity of the enzyme reaction. This is used to by-pass the reaction of tetrazolium reductases, mediating between the reduced coenzymes and the tetrazolium salt. However, if PMS is used, any active cytochrome oxidase must be inhibited, as it can either oxidize the PMS reducing the amount of formazan deposited at the site of the enzyme reaction or even reverse the pattern producing false non-selective staining and obscuring areas of recent myocardial damage.


Ronnie Houston, MS HT(ASCP)QIHC
Anatomic Pathology Manager

ChildLab, a Division of Nationwide Children's Hospital

www.childlab.com


700 Children's Drive
Columbus, OH 43205
(P) 614-722-5450
(F) 614-722-2899
ronald.houston <@t> nationwidechildrens.org<mailto:ronald.houston <@t> nationwidechildrens.org>
www.NationwideChildrens.org<http://www.NationwideChildrens.org>

"One person with passion is better than forty people merely interested."
~ E.M. Forster

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Laurie Colbert
Sent: Thursday, October 28, 2010 2:52 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Nitroblue Tetrazolium Chloride



I previously posted a question regarding Nitroblue Tetrazolium Chloride,

but I didn't really receive the info that I needed - so I thought I

would ask again.







I need to purchase this item for a research doc.  He wants to immerse

tissue in this solution for 24 hours and then process as usual.  It is

my understanding that this is some kind of dye.  I see that I can order

it from Sigma in tablet form.  I've seen it from other companies in a

powder form.







Has anyone ever used this reagent in the capacity that I describe?  Is

it available as a ready-to-use solution/liquid?  Is there a certain

strength/percentage that I should use?







Thanks,



Laurie Colbert



_______________________________________________

Histonet mailing list

Histonet <@t> lists.utsouthwestern.edu

http://lists.utsouthwestern.edu/mailman/listinfo/histonet


----------------------------------------- Confidentiality Notice:
The following mail message, including any attachments, is for the
sole use of the intended recipient(s) and may contain confidential
and privileged information. The recipient is responsible to
maintain the confidentiality of this information and to use the
information only for authorized purposes. If you are not the
intended recipient (or authorized to receive information for the
intended recipient), you are hereby notified that any review, use,
disclosure, distribution, copying, printing, or action taken in
reliance on the contents of this e-mail is strictly prohibited. If
you have received this communication in error, please notify us
immediately by reply e-mail and destroy all copies of the original
message. Thank you.