[Histonet] Buffy coat preparation for immuno

Stephen KumJew stevenk <@t> med.usyd.edu.au
Sun Nov 30 22:28:15 CST 2008

Hi from Down Under

    I was wondering if anyone had a method for preparing buffy coats for

What we have done so far

1) is spun down whole blood, extracted the buffy coat and made a
thrombin/plasma clot of the extracted buffy coat (minus RBCs). The clot was
post fixed overnight in 10% formalin and subsequently paraffin processed.
5um Sections were cut and immuno done. The clumping cells did not stain, but
the single ones on their own did.

2) To improve on fixation (assuming this was the problem with the unstained
clumping cells), the buffy coat was collected and fixed in 10% formalin. The
fixed buffy coat was washed in 0.9% NaCl twice, and then embedded in both  a
thrombin/plasma clot (unsuccessful), and  an agar block. The agar block was
paraffin processed. The subsequent immuno showed good staining in single
WBC, and in one clump of cells. The other clump showed no staining and had a
bit of cell damage.

    Sydney University

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