[Histonet] Bone decalcifying/processing problem

[Tony Henwood (SCHN)]

Orla,

You seem to have introduced many poor histologic processing practices into your technique.

Firstly ???4% paraformaldehyde. How did you make this up? The nomenclature is incorrect (see Histonet archives and Manoonkitiwongsa PS, Schultz RL. (2002) "Proper nomenclature of formaldehyde and paraformaldehyde fixatives for Histochemistry" Histochem J 34: 365-367).

Secondly, why fix and decal at 4oC. Low temperatures slow down the rate of diffusion and therefore fixation and can cause the formation of ice crystal artefact, which seems like what you have here.

Also if you are going to decalcify tissues then I would recommend doubling your fixation time. Remember one of the big advantages of aldehyde fixation is that it cross-links and thereby protects cell and tissue constituents from processing.

Try doing it all at room temperature, I bet that the artefacts will disappear.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Orla M Gallagher
Sent: Saturday, 13 July 2013 12:52 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Bone decalcifying/processing problem

Dear Histonetters,

Has anyone ever seen large holes/vacuoles in the bone marrow of mouse long bones after decalcification and processing to wax? We've had this problem with a study which was fixed in 4% paraformaldehyde and decalcified at 4 degrees C over a 14 day period according to a routinely-used protocol involving changes of 15% EDTA/0.5% PFA in PBS pH 8.0, rinsed in PBS to remove EDTA before processing to paraffin wax. The marrow is filled with large holes, almost like balloons. I'm happy to send images to anyone who might be able to help!

Best wishes,
Orla

--
**************************
Ms. Orla Gallagher
Bone Analysis Laboratory
Mellanby Centre for Bone Research
Department of Human Metabolism
D Floor Medical School
University of Sheffield
Beech Hill Road
Sheffield
S10 2RX
UK

Website: http://mellanbycentre.dept.shef.ac.uk

Tel:         0044114-2713337 (office)
              0044114-2713174 (lab)
E-Mail:    o.m.gallagher <@t> sheffield.ac.uk


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