[Histonet] alizarin red S mouse embryo alternate protocol question

[Nicole Collette]

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!
Nicole



  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 
specimens.

     PMID: 6165088 [PubMed - indexed for MEDLINE]