[Histonet] Chondrocyte loss in paraffin embedded cartilage sections

[Margie Teng]

   Hi,

   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]tengma <@t> orthosurg.ucsf.edu
   Research Assistant, Orthopaedic Surgery
   Veteran's Affair Medical Center, San Francisco and University
   (415) 221 - 4810 x 2743

References

   1. mailto:tengma <@t> orthosurg.ucsf.edu