Paraformaldehyde (pfa)

Harvested colons were washed with cold PBS, cut longitudinally, swiss-rolled and fixed with 4% paraformaldehyde (Junsei Chemical Co. Ltd., Tokyo, Japan). Paraffin sections were deparaffinized, rehydrated and stained with primary antibody against myeloperoxidase (Abcam, Cambridge, UK). After incubation with biotinylated secondary antibody, sections were developed using VECTASTATIN^® ABC kit (VECTOR Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin.

The colonies were scraped with a capillary glass tube (bent into a ring shape) and allowed to adhere onto 8-well chamber slides (chamber slide, NUNC, Waltham, MA). The next day chamber slides were rinsed twice with PBS, and the colonies were fixed in 2% paraformaldehyde (JUNSEI, Chuo-ku, Tokyo) for 20 minutes, washed twice with PBS, treated with 0.1% Triton X-100 for 5 minutes, rinsed twice with PBS, and treated with 2% BSA (Sigma Aldrich, St. Louis, MO)/PBS at room temperature for 1 hour. Thereafter, the colonies were treated with goat polyclonal anti-OCT4 or anti-hNANOG antibodies (diluted 1:1000 in 2% BSA/PBS, Abcam, Cambridge, MA) overnight at 4°C, washed twice with PBS, treated with Alexa Fluor 488 rabbit anti-goat IgG (Invitrogen, Carlsbad, CA) or Alexa Fluor 546 rabbit anti-goat IgG secondary antibodies (diluted 1:500 with 2% BSA/PBS) at room temperature for 1 hour. After rinsing with PBS three times, nuclei were stained with 300 nM DAPI (4,6-diamidino-2-phenylindole, Sigma-Aldrich, St. Louis, MO) at room temperature for 5 minutes in the dark. The colonies were washed with PBS three times, mounting medium (Vector Labs, Burlingame, CA) and coverslips were applied, and after 15 minutes, the cells were examined by a laser scanning confocal microscope equipped with Nomarski optics.

Cultured porcine ICM-derived cells were fixed in 4% (v/v) paraformaldehyde (Junsei Chemical Co., Japan). After two washes with Dulbecco’s phosphate-buffered saline (DPBS; Welgene), the fixed cells were stained with a solution containing 0.2 mg/ml napthol AS-MX phosphate (Sigma-Aldrich), 2% (v/v) N,N-dimethylformamide (Sigma-Aldrich), and 1 mg/ml Fast Red TR salt (Sigma-Aldrich) in 0.1 M Tris buffer (pH 8.2) for 90 min at room temperature. Subsequently, the stained cells were rinsed twice with DPBS, and the proportion of AP-positive cells was measured using a hemocytometer and an inverted microscope (CKX-41; Olympus, Japan).