Whole mount staining of cartilage and bone

Paul Z. Myers myers at astro.ocis.temple.edu
Thu Oct 27 07:12:27 EST 1994

In article <9409257831.AA783117593 at student.med.harvard.edu> ,
jfukuda at STUDENT.MED.HARVARD.EDU writes:
>    I'm a second year at Harvard Medical and wanted to know if there
were any 
>protocols out there that would allow efficient whole mount staining of
>and bone in zebrafish?

I've been doing a bit of this, using a protocol I got from Ruth
BreMiller at the University of Oregon. I'll repost the message 
she sent me summarizing the procedure:

> Tom Schilling uses this procedure:
> Fix fish one hour at RT in 10% neutral buffered formalin.
> Stain for a "short" overnight  (less for some mutants) in the following
>        10 mg Alcian blue
>        20 ml glacial acetic acid
>        80 ml 95% ethanol
>        (Filter before use)
> Rehydrate through the alcohols to DW
> Mount  (store) in glycerol
> Tom suggests that stained specimens can be dehydrated from the Alcian 
> blue solution and stored in methyl salicylate.
> Kris Vogel did some nice preps and her procedure is as follows:
> (She did not give me info about fixation but I would go with 10%
> formalin at RT for an hour)
> Alcian blue (15 mg/100 ml of 80% ethanol and 20% HAc) for 24 hr at RT.
> 2 to 5 hours in absolute alcohol (may be held here for up to 8 hrs.).
> 2 hours in Alizarin red (10 mg/100 ml of  0.5% KOH)
> 20% glycerol
> 50% glycerol
> Some additional thoughts:  Alcian blue is stable in organic solvents. 
I'm not 
> sure why an aqueous mount (glycerol) is used.  It apparently stains
> mucopolysaccharides at low pH's.   At high pH Alizarin red stains
> Again it seems to be stable in organic solvents.  I suspect that there
> problems with clearing, an especially sticky aspect of whole mount
> in general. 

I've used these procedures with good success (thanks, Ruth!) in larvae...I
haven't tried them on adults, and you'll probably need some revision to
them to work on big bony fish, if that's what you are trying to do. Alcian
blue and alizarin red are well-established, standard dyes, though, so you 
should be able to find more info in just about any book of histological

Paul Z. Myers                    myers at astro.ocis.temple.edu
Dept. of Biology                              (215) 204-8848
Temple University
Philadelphia, PA 19122

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