[Histonet] Chondrocyte loss in paraffin embedded cartilage sections
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.
Research Assistant, Orthopaedic Surgery
Veteran's Affair Medical Center, San Francisco and University
(415) 221 - 4810 x 2743
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