RE: [Histonet] Question on Von Kossa
That is how I do VK, I put it in the window for 20 min or so. I do a special HE counterstain on mine, using an aqueous eosin. The eosin will light up the osteoid by fluorescing under uv light. We use this with a image analysis system to measure total area, calcified bone area (light scope, from the black silver stain) osteoid seam thickness (fluorescent scope using the eosin fluorescent property, everything else will be dark except the osteoid seams) if you labeled the bone with a fluorchrome you can just look at an unstained section to measure area between two labels. Patsy -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Amos Brooks Sent: Friday, October 17, 2008 10:15 PM To: [EMAIL PROTECTED]; histonet@lists.utsouthwestern.edu Subject: [Histonet] Question on Von Kossa James, Try this: once the sections are brought to water, 5% silver nitrate in either bright sunlight or a 60-100 watt incandescent light bulb for 30 min (check for browning of the control tissue adjust time as needed). Rinse well in distilled water. 5% Sodium Thiosulfate for 1 min. Rinse and counterstain with nuclear fast red or whatever you think would look cool :-). Dehydrate, clear and coverslip the slides. Of course, as with any silver stain use acid cleaned glassware and gloves unless you like watching your fingers turn black in the sunlight. Really well decalcified tissue usually has the Calcium washed out. Try a calcified breast or something that wasn't decalcified. This causes microtomists to curse a lot, but it makes great VonKossa controls. Have fun, Amos Message: 10 Date: Thu, 16 Oct 2008 15:45:11 -0500 From: Herrick, James L. [EMAIL PROTECTED] Subject: [Histonet] Question on Von Kossa To: histonet@lists.utsouthwestern.edu Message-ID: [EMAIL PROTECTED] Content-Type: text/plain; charset=iso-8859-1 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 µm thick)? I would appreciate it greatly. Thank you much. Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Question on Von Kossa
James, Try this: once the sections are brought to water, 5% silver nitrate in either bright sunlight or a 60-100 watt incandescent light bulb for 30 min (check for browning of the control tissue adjust time as needed). Rinse well in distilled water. 5% Sodium Thiosulfate for 1 min. Rinse and counterstain with nuclear fast red or whatever you think would look cool :-). Dehydrate, clear and coverslip the slides. Of course, as with any silver stain use acid cleaned glassware and gloves unless you like watching your fingers turn black in the sunlight. Really well decalcified tissue usually has the Calcium washed out. Try a calcified breast or something that wasn't decalcified. This causes microtomists to curse a lot, but it makes great VonKossa controls. Have fun, Amos Message: 10 Date: Thu, 16 Oct 2008 15:45:11 -0500 From: Herrick, James L. [EMAIL PROTECTED] Subject: [Histonet] Question on Von Kossa To: histonet@lists.utsouthwestern.edu Message-ID: [EMAIL PROTECTED] Content-Type: text/plain; charset=iso-8859-1 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 µm thick)? I would appreciate it greatly. Thank you much. Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[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 µm thick)? I would appreciate it greatly. Thank you much. Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Question on Von Kossa
Jim, 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. Jack Date: Thu, 16 Oct 2008 15:45:11 -0500 From: [EMAIL PROTECTED] To: histonet@lists.utsouthwestern.edu 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 µm thick)? I would appreciate it greatly. Thank you much. Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _ Want to read Hotmail messages in Outlook? The Wordsmiths show you how. http://windowslive.com/connect/post/wedowindowslive.spaces.live.com-Blog-cns!20EE04FBC541789!167.entry?ocid=TXT_TAGLM_WL_hotmail_092008___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
