Staining

Paul Z. Myers myers at astro.ocis.temple.edu
Sun Mar 17 13:26:06 EST 1996


In article <Pine.SOL.3.91.960315111656.21485B-100000 at soc1.acpub.duke.edu>,
ylin at ACPUB.DUKE.EDU (Yiing Lin) wrote:

>Hello,
>
>Take a look at:
>
>  Stachel, S.E., D.J. Grunwald, and P.Z. Myers (1993) Lithium 
>  perturbation and goosecoid expression identify a dorsal specification 
>  pathway in the pregastrula zebrafish. Development 117:1261-1274. 
>
>They stained the notochord with an antibody (MZ15).
>
>I'm not sure about cartilage staining; would alizarine red or alizarine 
>complexone do the job?
>
>                                                -Yiing Lin
>                                                 ylin at acpub.duke.edu
>
>On 15 Mar 1996, James Bottesch wrote:
>> I was wondering if anyone out there knows of an effective staining 
>> protocol for the notochord in developing embryos.  Also, if you know of a 
>> protocol for effectively staining cartilage, it would be a great help.

I've been using a protocol I got from Ruth BreMiller at UO, who in turn
credits Tom Schilling and Kris Vogel. Here's the recipes straight from Ruth:

--------

Tom Schilling used this one for fish at least 3 days old:
Fix fish one hour at RT in 10% neutral buffered formalin.
Stain for a "short" overnight  (less for some mutants) in the following solution
       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% buffered  
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

--------

We've only used Tom's protocol, since it worked well the first time we tried
it.

-- 
------------------------------------------------------------
Paul Z. Myers                   myers at astro.ocis.temple.edu
Dept. of Biology                myers at netaxs.com     
Temple University               http://fishnet.bio.temple.edu
Philadelphia, PA 19122          (215) 204-8848




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