Rabbit bone labels

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From:Manuel J Jayo <jayopai@mindspring.com>
To:'HistoNet Server' <histonet@pathology.swmed.edu>, larisonk@uoneuro.uoregon.edu, histohappens@hotmail.com, Mstevens@genetics.com, uvsgc@msu.oscs.montana.edu
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Responding to the answers already provided below. all these are good
suggestions if you are trying to label the mineralization of osteoid or
fracture repair tissue, in order to conduct histomorphometric measurements.
As Mary indicated, there are several options and you will need different
filters in your microscope setup for different labels to be excited by the
UV and to be able to detect fluorescence.

As a veterinarian, just two words of caution. First, rabbits (or their gut
bacterial flora) may be sensitive to tetracycline. Although tetracycline
(demeclocycline) has been used in several papers, you should contact your
veterinarian in house when giving any amount. Secondly, hypocalcemia. If you
are to give any of the fluorochromes intravenously as a bolus, you may cause
acute hypocalcemia (low blood calcium) and kill the animals. In my
experience, I have seen this happen more with xylenol orange. The reason is
that the fluorochromes may chelate some of the circulating calcium.

Dose ranges to keep in mind for tetracycline and calcein for most species
are from 10 to 25 mg/kg. Always keep every fluorochrome in the dark or it
will become useless.

Calcein (to make a 10 mg/ml solution)
Making 40 ml
Calcein (Sigma #C-0875)         400 mg
NaHC03 (Fischer #5233-500)      400 mg
Sterile Saline                  40 mls
Stir covered until dissolved.
Use sterile filter and aliquot into the sterile clot tubes. Keep in dark
(away from UV) and refrigerate until ready to use (remove 1/2 hr before use
for room temp). Do not keep longer than 1 week.

Demeclocycline (tetracycline)
Prepared each afternoon before labeling. Takes longer to dissolve.
Demeclocycline (Sigma #D6140) (7-chloro-6-demethyltetracycline)
A rat example, to be given at 15 mg/kg. If you have 14 rats and each rat
weighs 300 g
14 x 300 = 4.2 kg x 15 mg/kg = 63 mg required, get approximately 70 mg in 7
ml to have a 10 mg/ml sol. A 0.300 Kg rats gets 4.5 mg or 0.45 ml.

I have enclosed some references that may be of help. Wish you luck.

Manuel J. Jayo DVM, PhD, DACVP
Senior Pathologist
Pathology Associates International
119 Highway 801 South
Suite A-300
Advance, NC 27006
336.998-3021 (fax)

Busa R, et al. Neovascularized bone grafts: experimental investigation.
Microsurgery.  19(6):289-95, 1999. Tetracycline injection.

Konig Junior B, et al. A study of different calcification areas in newly
formed bone 8 weeks after insertion of dental implants in rabbit tibias.
Anatomischer Anzeiger.  180(5):471-5, 1998
The bone tracers used were Alizarin, Calcein and Xylenol-orange.

Boyde A, et al. Vital confocal microscopy in bone. Scanning.  17(2):72-85,
1995. A variety of intravenously administered substances, including
fluorescein, fluorescein-dextrans, fluorescent microspheres, acridine
orange, DASPMI, calcein, and tetracycline.

Bogoch E, et al. Healing of cancellous bone osteotomy in rabbits--Part I:
Regulation of bone volume and the regional acceleratory phenomenon in normal
bone. Journal of Orthopaedic Research.  11(2):285-91, 1993. During the
recovery period, the rabbits received the bone label calcein in their
drinking water.

Bogoch E, et al. Healing of cancellous bone osteotomy in rabbits--Part II:
Local reversal of arthritis-induced osteopenia after osteotomy. Journal of
Orthopaedic Research.  11(2):292-8, 1993. Used intravital bone label

Tsai CL, et al.  Additive effects of prostaglandin E2 and pulsed
electromagnetic fields on fracture healing. Chinese Journal of Physiology.
34(2):201-11, 1991.
Bone formation of fractured fibulae assessed with fluorescent labelling
compounds, tetracycline, xylenol orange, and calcein.

Volpon JB, et al. The use of decalcified granulated homologous cortical bone
matrix in the correction of diaphyseal bone defect. An experimental study in
rabbits. Archives of Orthopaedic & Traumatic Surgery.  99(3):199-207, 1982.
New bone formation was followed by histology and incorporation of calcein
blue, xylenol orange and tetracycline.

Sudmann E and Bang G. Indomethacin-induced inhibition of haversian
remodelling in rabbits. Acta Orthopaedica Scandinavica.  50(6 Pt 1):621-7,
1979. Rabbits were given calcein and oxytetracycline fluorochrome labels.

Rahn BA and Perren SM. Calcein blue as a fluorescent label in bone.
Experientia.  26(5):519-20, 1970. Compared to tetracycline.

Date: 28 Jul 2000 10:59:07 -0500
From: "Mary Stevens" <Mstevens@genetics.com>
Subject: Re: fluorescent labeling bone

Hi Susan,

There are several options available - here they are

Calcein #C-0875 - Sigma Co. Excitation @ 420-490, SQ injection @ 10mg/kg -
give this as your term labels - approximately on 13 and 3 days prior to
sacrifice.  If you suspect that your rabbits have severally depressed bone
formation (including your controls) you can give it on 2 consective days
~14/13 and 4/3 prior to sac.

Tetracycline # T3383, Sigma Co., Excitation @ 380-420, SQ @25mg/kg injection
at day 0 of study.

Don't give the Cal and Tet as the term labels together, the cal is pretty
bright, and can shadow the tet.

Another option, not as bright or easy to read against the dark bone, is
Alizarin Red S, Sigma Co # A5533 SQ @ 30mg/kg.  This could be used in place
tet, but I wouldn't recommend it in place of the calcein.

Good luck,

Date: 27 Jul 2000 22:24:17 -0500
From: Karen Larison <larisonk@uoneuro.uoregon.edu>
Subject: Fwd: fluorescent labeling bone

Someone here wanted to label growing bone in the operculum of baby
fish.  I haven't tried it myself, but found this reference:
"Staining of the calcification front in human bone using contrasting
fluorochromes in vitro" J Histochem Cytochem 32, 1251 (1984).

Karen in Oregon

>Date: Thu, 27 Jul 2000 07:48:37 -0500 (CDT)
>From: susan king <histohappens@hotmail.com>
>Subject: fluorescent labeling bone
>To: Histonet@pathology.swmed.edu
>Good Morning!
>     We want 2 label bone in rabbits and as I'm sure U all know..they
>want it yesterday.  Any recommendations as to what labels to use and
>where to get it would B greatly appreciated. Seems that old
>suppliers have dried up for injectables.
>                Thank You! Susan
>S. King
>Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com


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