Nuclear staining with DAB

From:Dave Lizamore

Hi All,
We are trying to detect Y-chromosomes in mouse tissues using in situ
hybridisation. I use  FITC and biotin labeled probes. from Cambio. The
FITC probes works very well with little or no background, but the biotin
probe is driving me up the wall. I use Vectastain's ABC kit with DAB to
visualise. Irrespective of the tissue type, between 50-80% of the nuclei
stain with the DAB. There two distinct type of nuclear staining visible.
Some nuclei display an intense granular stain in the vicinity of the
nuclear membrane, while others stain a homogeneous light brown. The
cytoplasm of all the cells are perfectly clear with no background. I
incubate for 10 min in PBS/Tween/Milk powder at 37 degrees C followed by
30 min in 0.3% H2O2 in methanol prior to ABC application. Both PBS and
SSC are at pH 7.0 and ABC are at pH 7.5 as the manufacturer recommends.
Any suggestions would appreciated.
Dave Lizamore     (
Faculty of Health Sciences  School of Anatomical Sciences
University of the Witwatersrand
Tel +27 (011) 717 2716   Fax: +27 (011) 717 2702

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