Re: [Histonet] alizarin red S mouse embryo alternate protocol question

From:Nicole Collette

Hello, Tamara,

The protocol calls for 1% KOH. I could drop it down, but I was also 
concerned about osmotic issues with transitioning from 95% ethanol 
straight into full aqueous solution. I have done the protocol with 
E17.5 embryos (bone and cartilage), and they survive just fine, I 
basically stain in 1% KOH O/N and skip all other KOH steps to avoid 
overdigestion of those little guys. When I tried it with these 
younger guys they looked OK after staining for a few hours (not O/N) 
except for the heads exploding, and then during subsequent clearing 
in glycerol (no KOH) they fell apart. Thanks for your advice and help.


>what percentage of KOH are you using. we use trypsin for our digestion
>step and fix in buffered formalin or paraformaldehyde. I routinely stain
>chicken embryos for bone and/or cartilage.
>Tamara Franz-Odendaal (PhD)
>Assistant Professor, Biology, MSVU
>Mount Saint Vincent University
>166 Bedford Highway
>Halifax, NS,  B3M 2J6
>Tel: +1 902 - 457 6140 (office)
>Tel: +1 902 - 457 6718 (lab)
>Fax: +1 902 - 457 6455
>>>>  Nicole Collette  09/13/07 4:20 PM >>>
>Hello, All,
>I am trying to do skeletal stains with whole mouse embryos
>E14.5-E16.5 with alizarin only to look at gross mineralization of
>bones (the alcian blue interferes with interpretation since the
>cartilage and bone overlay each other, and this experiment should be
>straightforward enough not to require sectioning). I have tried this
>stain with a similar protocol to what we use for adults, which calls
>for alizarin in KOH, followed by KOH digestion and clearing. When I
>tried this on that same ages of embryos recently, I got heads that
>exploded, presumably due to some osmotic issue (happened during
>staining, not during subsequent digestion/clearing), and despite
>watching the embryos very carefully not to overdigest, they
>overdigested and were essentially useless. I found the Arnott
>protocol in the Atlas of Mouse Development, that calls for fixation
>in ethanol, dehydration/lipid dissolving in acetone, staining in
>ethanol, then KOH. When I put the alizarin in acid alcohol, I get a
>yellow solution (that dissolves poorly in ethanol, too), is KOH
>required to make it then turn red/purple? Would it be better to try a
>more gradual way to negotiate the change from 95% ethanol to 1% KOH?
>I also found a modification of this protocol that doesn't use KOH at
>all, but it's in a journal I can't get my hands on, reference is
>below. I am concerned that if I stain in acid alcohol,
>dehydrate/clear in ethanol:glycerol instead of KOH/glycerol, I won't
>get a color change. Perhaps some combination of high pH/ethanol would
>be better? Any help would be most appreciated...
>Thanks in advance for the help!
>   Teratology. 1980 Dec;22(3):299-301.Links
>      Differential staining of cartilage and bone in whole mouse
>fetuses by alcian blue and alizarin red S.
>      McLeod MJ.
>      The procedure described by Inouye ('76) for the staining of
>full-term mouse fetal skeletons has been adapted for use with mouse
>embryos and fetuses of days 14-18 of gestation. The main adaptations
>for younger specimens involve a longer time in acetone, in lieu of
>skinning, and omission of the aqueous KOH step. These adaptations
>require more time but result in consistently good staining of intact
>      PMID: 6165088 [PubMed - indexed for MEDLINE]
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