Here you go:
MMA Thin (5 microns) Sections
1) Deplastify with three changes of Xylenes @ 60C for 30-60 minutes (depending on size of section). You can also use three changes of acetone @ RT for 15-30 minutes.
2) Hydrate tissue from ethanol to DI water (100% EtOH for 5 min, 95% EtOH for 5 min, 70% EtOH for 5 min, DI H2O for 5 min) @ RT.
3) Stain in 5% Silver Nitrate solution @ RT for 5 minutes. Keep tissue and solution protected from light!!!!
4) Three DI water rinses @ RT for one minute each.
5) Develop all bound silver ions in Sodium Carbonate-Formaldehyde solution (5 g sodium carbonate, 25 mL formaldehyde, 75 mL DI water) @ RT for 2 minutes (TIME CRITICAL).
6) Two DI water rinses @ RT for one minute each.
7) Stop reaction and remove all unbound silver ions in Farmer's Diminisher (20 g sodium thiosulfate, 1 g potassium ferricyanide, 210 mL DI water) @ RT for 30 seconds. This solution is only stable for 30-45 minutes so make and use fresh.
8) Wash in running tap water for 20 minutes.
9) Rinse in DI water @ RT for one minute.
10) Counterstain with 5% MacNeal's tetrachrome solution. There are other choices, but this is what I use.
11) Dehydrate to Xylenes and coverslip.
I will send you a copy of my detailed protocol.
> Date: Thu, 16 Oct 2008 15:45:11 -0500> From: Herrick.James@mayo.edu> To: firstname.lastname@example.org> Subject: [Histonet] Question on Von Kossa> > Hello everyone,> > Does anyone have a good Von Kossa stain protocol that they would not mind sharing, on animal bone tissue (femur/tibia) that has been embedded in GMA or MMA (sections are between 5 and 10 Ám thick)? I would appreciate it greatly. Thank you much.> > Jim> > > > _______________________________________________> Histonet mailing list> Histonet@lists.utsouthwestern.edu> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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