Re: [Histonet] Chondrocyte loss in paraffin embedded cartilage sections
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
> Research Assistant, Orthopaedic Surgery
> Veteran's Affair Medical Center, San Francisco and University
> (415) 221 - 4810 x 2743
> 1. mailto:firstname.lastname@example.org
>Histonet mailing list
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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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