Anti rabbit igg hrl f ab 2 pe

Cells were collected from each medium type at days 7, 15, and 20. Cells were washed in phosphate-buffered saline (PBS) and immediately fixed in 10 % formaldehyde solution. Each cell sample was exposed to CD133, CD90, E-cadherin, and beta-catenin primary antibodies. After a 24-h incubation, the anti-rabbit IgG HRL-F (ab) and 2-PE Anti-goat IgG-FITC (Santa Cruz, TX, USA) secondary antibodies were attached. Antibodies are described in Table 2. Immunostained cells were observed with a fluorescence microscope.Table 2

Antibodies used for immunocytochemistry

First antibody (1:300 dilution)	Secondary antibody (1:300 dilution)
Rabbit anti-human E-cadherin antibody (Santa Cruz)	Anti-rabbit IgG HRL-F(ab)2-PE (Santa Cruz)
Rabbit anti-human CD90 antibody (Santa Cruz)	Anti-rabbit IgG HRL-F(ab)2-PE (Santa Cruz)
Goat anti-human beta-catenin antibody (Santa Cruz)	Anti-goat IgG-FITC (Santa Cruz)
Goat anti-human CD133 antibody (Santa Cruz)	Anti-goat IgG-FITC (Santa Cruz)

For confocal microscopy, cells were fixed in glass chamber slides with 10 % paraformaldehyde in PBS for 30 min at room temperature. After two washes with PBS, cells were permeabilized with 100 % methanol for 5 min at room temperature. Cells were washed twice with PBS and blocked for 45 min in 5 % bovine serum albumin (BSA) in PBS. Confocal microscopy studies were carried out using a laser-scanning microscope from OLYMPUS (Tokyo, Japan).