[Histonet] Safranin O

[Jack Ratliff]

Have you tried Sanderson's Rapid Bone Stain? Try using it first (7 minutes at 60C), then rinse in dH2O (a few dip an dunks at 60C and blot dry), then counterstain with safranin O for 2-5 minutes (room temp - check intensity) and rinse in 100% EtOH (room temp - few dip and dunks and blot dry). If saf o is too intense, you should be able to differentiate a little with 95% EtOH first then do the 100% EtOH step.

On a curious note, is there any particular reason why you are cutting thick sections and grinding? You should very easily be able to cut thin sections with a rotary microtome using a tungsten-carbide knife at 5 microns and then you would have greater staining flexibility. Call me if you want to discuss (317-281-1975).

Jack

On Feb 2, 2011, at 4:18 PM, "Herrick, James L. (Jim)" <Herrick.James <@t> mayo.edu> wrote:

> Hello everyone,
> 
> I am trying to stain for cartilage in mouse skull (cross-sectioning with
> the brain in tact) and wanted to know if anyone might have a good
> protocol for Safranin O / Fast green or Safranin O / von Kossa? The
> tissue is embedded in MMA and was cut on a saw microtome and finished
> down to an approximate 20 to 30 micron tissue thickness - 50 microns
> including the glue layer (I glue a slide to the block before making the
> cut and then grind and polish to finish). I tried Weigert's hematoxylin
> / Safranin O / Fast Green ( I began with Weigert's Hematoxylin, followed
> by Fast Green and finished with Safranin O), but the Safranin O seemed
> to overpower the Fast Green (the calcified bone stained more red than
> green). Any help on this issue would be greatly appreciated as always.
> Thanks again.
> 
> Jim
> 
> 
> 
> 
> 
> 
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