[Histonet] Von Kossa staining on PMMA sections

[Jack Ratliff]

Paul,

 

For MMA embedded specimens (MMA +DBP), I first deplastify my sections, hydrate to water, stain in 5% silver nitrate solution for 5 minutes (in the dark), wash times three changes in DI H2O (in the dark), develop in sodium-carbonate formaldehyde solution for 2 minutes (in the dark), wash times two in DI H2O (back under normal lighting conditions), then stop the reaction in sodium thiosulfate + potassium ferricyanide solution for 30 seconds, and immediately rinse in running tap water for 15 minutes.

 

The Von Kossa reaction results from process above then yields black mineralized bone. After the tap water rinse, I generally counterstain with 2% MacNeal's tetrachrome for 5 minutes, rinse in DI H2O, and dehydrate to xylenes to coverslip. This then reveals immature bone formation or osteoid = grayish or jaded green, growth plate cartilage = purple, osteoblasts = blue, osteoclasts = blue-green, bloods cells = greenish, etc.

 

Feel free to contact me if you have any additional questions.

 

Jack


 
> Date: Thu, 12 Mar 2009 11:19:49 -0400
> From: PMonfils <@t> Lifespan.org
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Von Kossa staining on PMMA sections
> 
> The standard Von Kossa silver stain for calcium calls for 20 minutes in the silver nitrate solution under UV light. There is a modified Von Kossa for plastic embedded bone sections, which is identical except it calls for a minimum of 6 hours in the silver nitrate solution under UV. Does anyone know why such a long staining time is recommended? Visually the calcium in the bone sections turns black within 20 minutes, so why is so much additional time needed? Thanks.
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