Re: [Histonet] Chondrocyte loss in paraffin embedded cartilage sections

From:Gayle Callis

 From what you describe, the chondrocytes are probably mechanically 
displaced from their lacunae in the cartilage during the sectioning 
step.  Is there bone attached to the cartilage?  and what size are the 
samples and what is your processing schedule?

Cartilage tends to be drier than surrounding bone (you did not say if any 
was attached) due to its high water content, so during processing, more of 
the bound water is removed along with free water in tissue.  After trimming 
the block, soak it on ice block with water briefly.   Use a brand new 
disposable blade, preferably high profile to insure stability, but low 
profile will work IF the cartilage is perfectly processed which could 
entail longer schedule with thicker samples.  Some disposable blades work 
better with cartilage/bone samples, and change to a fresh sharp edge (even 
between ribbons!) frequently to insure the cartilage is perfectly flat when 
sectioned.  What you see, with nuclei askew in tissue and dried into 
place/adhered to slide happens before the staining.

Also, if the cartilage is perfectly processed with total infiltration by 
paraffin - the latter will support all structures better to avoid 
displacement of nuclei.

M 12/17/2004, you wrote:

>   I  am trying to perform immunohistochemistry assays on human cartilage
>   sections.  I have a problem with chondrocyte loss - when I look at the
>   DAPI  stains,  I  see  a  significant  number of nuclei outside of the
>   boundaries  of the tissue.  There are a lot of empty lacunae, although
>   I think that is also related to the initial quality of the tissue.
>   Does anyone have any suggestions to prevent this from happening?  I am
>   using  Fisherbrand Superfrost Plus microscope slides.  The assays that
>   I  have  observed this with are: TUNEL, In Situ Oligo Ligation (ISOL),
>   and an assay for single-stranded DNA using mouse monoclonal antibody.
>   I  rehydrate  my samples using three changes of xylene, two changes of
>   100%  ethanol  (etOH),  1x  95% etOH, and 1x 70% etOH.  I am trying to
>   compare the difference between incubating my samples for 10 min versus
>   15  min  in  20  microgram/mL  proteinase  K,  but  I  am worried that
>   decreasing my proteinase K incubation might mask my DNA.
>   I can provide further details if needed.
>   Thank you,
>   Margie Teng
>   ------------------------------
>   Margie Teng
>   [1]
>   Research Assistant, Orthopaedic Surgery
>   Veteran's Affair Medical Center, San Francisco and University
>   (415) 221 - 4810 x 2743
>   1.
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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