Re: osteiod stain

From:Gayle Callis <>

You should be able to observe osteiod on an H&E slide, Movat's pentachrome
probably works also.  There are other methods in the literature, but never
used them, more complicated and didn't fit into the protocol for other
special stains.  There is not doubt undecalcified plastic (PMMA) embedded
bone is superior for observing osteoid, mainly because the bone remains
calcified next to the osteoid.  Sanderson rapid bone stain is designed for
use on plastic sections and NOT FOR PARAFFIN.  

For paraffin sections, you need control decalcification using endpoint
determinations which helps maintain good staining for nuclei and other
tissue components.  Must must be totally fixed, NBF works great, decalcify,
test, rinse bone with water to remove acid.  For staining, you may extend
hematoxylin staining to insure good nuclear staining and also bring out
other features in bone.  Lamellae and canaliculi can be seen with H&E stain. 
Stain in hematoxylin for 10 min rather than 2 or 3, Erhlichs hematoxylin is
excellent for bone and cartilage, Harris will work well if fresh and
eliminate the differentiation step entirely, just blue, rinse the bluing
out for 1 - 2 min, go to alcohol before eosin/phloxine.   

Use eosin phloxine (Richard Allan now sells this, I believe) for
counterstain and back off the time.  10 dips may suffice, rinse well in 95%
(up to 3 changes) continue on. 

Surprising how you can make an H&E stain work for osteoid, it takes some
tweaking. Most problems arise from understaining by hematoxylin,
overstaining with eosin/phloxine. 

Also try 1% toluidine blue, 1 min at RT, rinse in tap water for a minute,
AIR DRY SLIDES, then mount.  Do not go through alcohols UNLESS you want to
remove stain, 95% doing most of the work, 100% removes less.  You can back
off staining time if too dark, this is the easiest stain to do. 

 Movats pentachrome should also work with some methods long!  J of
Histotechnology has some modified, shorter versions.  For the Safranin du
gatinais (sp?), go to grocery store and buy saffron in spice section,
cheaper and works.  

Have fun,


At 08:14 AM 4/24/01 -0500, you wrote:
>We are using paraffin embedded blocks.
>>>> Gayle Callis <> 04/23/01 09:52AM >>>
>What is the embedding, paraffin or polymethylmethacrylate?  
>Several ways to do this
>At 03:46 PM 4/20/01 -0500, you wrote:
>>Hello histonetters,
>>Does anyone out there have any experience with a tetrachrome method for
>osteiod?  If so, I'm anxious to hear your replies!
>>Renee Escalona, BS, HTL(ASCP)
>>Laboratory Supervisor
>>TX. Vet.  Med. Diag. Lab.
>>College Station, TX
>Gayle Callis
>Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>Bozeman MT 59717-3610
>406 994-6367
>404 994-4303 (FAX)
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)

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