cartilage lift up, mouse Re: [Histonet] section edges lifting

[Gayle Callis]

If one tries to flatten cartilage with a brush after it comes out of 
clearing agent, the cartilage often tears rather than flattening - been 
there, done that way too many times.   It could be the cartilage is too dry 
to begin with, as it has a high content of water and overdehydration during 
processing may be a culprit.  Check your processing schedule, in general, 
mouse knee joints will be totally processed in either 1 hour per station to 
1 1/2 hours per station starting with 70%, 80% 95% X 2, 100% x 2, xylene x 
2 and 3 paraffin changes at 60C using vacuum and pressure, automated 
processing is a huge help.

If you have to make the cartilage lay down ,  it is better to do that after 
a water rinse and before alcohols/clearing agents in dehydration as 
solvents only make this tiny strip of articular cartilage brittle.   It 
also helps to soak the trimmed bone block on ice with water on top before 
you start sectioning, just don't soak so long that the tissue swells out of 
the block and use the sharpest blade you have.  The sections must be 
totally flat, compression free when picked up on Plus charge slides.

After picking up on Plus charge (silane) we drain slides for 15 min or so, 
and then ALWAYS dry bone sections FLAT, at 37C to 40C to ensure the 
sections stay down.  Flat drying in an oven is done over a period of days, 
36 to 48 hours might be a ball park for time in terms of days,  rather than 
overnight.  This is for murine knees, paws, ankle joints, skulls, tibias, 
femurs and we do experience cartilage lift up. Gentle rinsing is a must.



  At 07:18 AM 9/27/2005, you wrote:
>Check on the expirartion date of the slides. After some months they don't 
>work as they are supposed to. Slides should be drained/dried on edge (not 
>flat). A last resort would be to coverslip by hand, looking at the edges 
>(and even flattening them out with a hair brush before coverslipping).
>Rene J. Buesa
>
>Sam Vaughn <samvaughnhisto <@t> gmail.com> wrote:
>I'm new to histonet and have a question about histo staining.
>I have 6 um paraffin sections of mouse knee joints on uncoated superfrost
>plus slides. I collect the sections from a distilled water bath and let them
>drain at least 30min before placing them at 37 degrees overnight before
>staining. After Safranin O/ Fast Green staining a very thin area around the
>edge at the edge of the tissue lifts off the slide rendering it impossible
>to visualize with the microscope. Of course this is the the articular
>surface which we are interested in! I've used the same slides in the past
>without any coating and it has worked fine. I'm in a new lab now and trying
>to get things running. Any help you could give me would be really
>appreciated!
>Thanks,
>Sam
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)