Felicity:

I have recently investigated various staining techniques for evaluating bone
histology using a Zeiss Axiovert CSLM. I have tried two conventional stains
and one modified stain we use for our applications. And have compared these
to immunofluorescent antibodies to individual bone cell markers. I can send
you by fax or snail mail those procedures if you like.

Briefly, the standard histology stain procedure [found in any histology
text] called H & E (Hematoxylin and Eosin) worked well due to the
autofluorescent nature of eosin. The second standard histology procedure is
a May-Grunwald Geimsa (using Jenner's stain and Geimsa stain, the procedure
is found in Armed Forces Institute of Pathology Manual by Lee Luna 3rd ed.
page 121- 122). These polychromatic stains contain several autofluorescent
dyes. These two stains will allow you to compare directly with DIC or
transmitted light microscopy.

For our purposes, investigation of bacterial adhesion in bone, we use a
modified Gram's stain procedure which we developed and published. The
crystal violet which stains gram positive bacteria is polychromatic and
autofluorescent. The secondary staining with Van Gieson assists dramatically
with morphology. I can email you examples of each taken on our CSLM off the
list (since the files are quite large.)

Hope this helps. Let me know if you want our modified Gram stain protocol or
the pictures.

Regards,

Donna Montague
Research Assistant
Orthopaedic Research
University of Arkansas for Medical Sciences
Little Rock, Arkansas, USA
> ----Original Message-----
> From: Felicity Lawrence [SMTP:[log in to unmask]]
> Sent: Tuesday, September 22, 1998 9:20 PM
> To:   [log in to unmask]
> Subject:      Confocal of bone
>
> Dear all,
>
> I am fairly new to this discussion group so I apologise in advance if this
> topic has been covered in detail already.
>
> Basically, I have a post-grad student who would like to assess the
> architecture of bone (compact and spongy).  She is looking for a
> fluorescent
> dye that will enable imaging.  She doesn't need to disctiminate between
> the
> different cell populations but rather is looking at the arrangement of
> trabeculae, etc.  We have a krypton/argon laser with 488nm, 568nm and
> 647nm
> excitation.  We have a number of filters available for detecting emmision
> on
> our Leica TCS4d CLSM.
>
> Can anyone help me out with a dye please?
>
> Many thanks,
> Felicity
>
> Felicity Lawrence
>
> Analytical Electron Microscopy Facility
> Queensland University of Technology
> GPO Box 2434, Brisbane 4000
> Australia
>
> Ph : 3864 2557
> Fax: 3864 5100