Abiorevo bz 9000 microscope

Peripheral blood samples were collected from the inferior vena cava of anesthetized mice
by using a syringe containing EDTA-2K solution. Blood count was determined with an
automated hemocytometer (Nihon Kohden, Tokyo, Japan). Blood samples were smeared onto
microscope slides and stained with May-Grünwald-Giemsa stain, and then photographed with a
BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan).

A homozygous GalT/CMAH double KO pig (M129-2), a homozygous
GalT KO pig (M71-5) and an age-matched wild-type (WT) pig were sacrificed 2 days after
birth. Harvested samples were used for immunohistological analysis of the H-D antigen. The dissected organs
were fixed in a 4% paraformaldehyde solution (Wako Pure Chemical Industries, Osaka, Japan), embedded in
paraffin, sectioned and stained with hematoxylin using standard methods.
The fixed sections were also incubated with blocking solution (2% BSA/D-PBS) for 1 h and then treated with a
chicken anti-H-D antibody (a generous gift from Prof N Wakamiya, Asahikawa Medical University) for 1 h. After
removal of the excess antibody, the sections were incubated with biotin-conjugated goat anti-chicken IgY
(Abcam, Cambridge, United Kingdom) for 30 min. For detection of the α-Gal antigen, the sections were incubated
for 1 h with biotin-conjugated Griffonia simplicifolia I (GS-IB4) lectin (Life Technologies).
Each section was also incubated with Streptavidin-Horseradish Peroxidase (HRP) (DAKO, Tokyo, Japan) for 15 min
and 3,3′-Diaminobenzidine tetrahydrochloride (DAB) (DAKO) for 5 min. The slides were visualized using a
Biorevo BZ-9000 microscope (Keyence, Osaka, Japan).