[Histonet] TRAP Staining

[Silvina Molinuevo]

Dear Dave, we use the next protocol with pretty good results. 


For diazotation reaction (Solution A):
Dissolve 7mg  Fast Garnet GBC base in 1ml of methyl or ethylenglygol. Add 1ml HCl 0.1N. Add drop by drop 2.07mg NO2Na. Keep T below 15ºC.

Solution B:12.5mg/ml Naftol ASBI phosphate in distilled water

For the enzymatic reaction: 

Disolve 75mg Sodium tartrate in 45 ml Citrate buffer pH 4.9. Pre-warm to 37ºC

Add

2ml Solution A
0.5ml Solution B

Incubate the slides about 1h at 37ºC.


The most difficult thing is the diazotation reaction. 

You must have the stoichiometric relationship between Fast Garnet and Sodium nitrite. Sodium nitrite must be as pure as you can. You also have to retain the stoichiometry relation with chlorhidric acid to avoid reaction subproducts. Diazotation T must be not over 15ºC and you should use the diazotation product 


Best regards, silvina


Dr María Silvina Molinuevo
Grupo de Investigacion en 
Osteopatias y Metabolismo Mineral
Departamento de Ciencias Biologicas
Facultad de Ciencias Exactas
Universidad Nacional de La Plata
47 y 115
(1900)La Plata
Argentina
www.biol.unlp.edu.ar/giomm
e-mail: silvina.molinuevo <@t> bigfoot.com