Ultrakitt

Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a series of graded alcohols. After incubation of 3% H₂O₂ in PBS with 0.1% Triton X-100 at room temperature for 30 minutes, the sections were blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour. The sections were incubated with a rabbit polyclonal anti-GALNT2 antibody (Sigma-Aldrich) at 1:200 in 1% BSA/PBS at 37ºC for 16 hours. After rinsing twice with PBS, the Super Sensitive Link-Label immunohistochemistry Detection System (BioGenex) was applied to tissue sections. Specific immunostaining was visualized with 3,3-diaminobenzidine liquid substrate system (Sigma-Aldrich). All sections were counterstained with hematoxylin and mounted with UltraKitt (J.T. Baker). For negative controls the primary antibodies were replaced with a control non-immune IgG at the same concentration. The immunoreactivity of GALNT2 was examined in a blinded manner by two independent investigators (Dr. Hsu WM and Dr. Jeng YM) without knowledge of clinical background of the patients. GALNT2 expression levels in NB tumors examined by immunohistochemical staining correlated well with those examined by Western blotting, as described in our previous study [22 (link)].

Chromosome spreads were prepared from corn snake fibroblasts established from an E30 embryo. Fibroblasts were grown at 30 °C in MEM (M5650; Sigma-Aldrich) supplemented with 2 mM L-glutamine (no. 25030024; Gibco), 10% calf serum (C8056; Sigma-Aldrich), 100 U/mL penicillin/streptomycin (no. 15140122; Gibco), 2.5 μg/mL amphotericin B (A2942; Sigma-Aldrich), and 50 μg/mL gentamicin (no. 15750037; Gibco). KaryoMAX Colcemid Solution in PBS (no. 15212012; Gibco) was used at 0.1 μg/mL to arrest cells in metaphase. Cells were collected after trypsin digestion and incubated for 10 min at 37 °C in a 0.075 M KCl solution. They were fixed in methanol:acetic acid (3:1), and the cell suspensions were dropped onto clean glass slides and air-dried. Chromosomes were stained with KaryoMAX Giemsa Stain Solution (no. 10092013; Gibco), rinsed with distilled water, air-dried, and mounted with Ultrakitt (no. 3921; JT Baker).

Dorsal and ventral skin was fixed in 4% PFA and dehydrated in ethanol before embedding in paraffin blocks. Seven-microgram microtome sections were deparaffinized and directly mounted with Ultrakitt (no. 3921; JT Baker) to image the chromatophores with a Pannoramic MIDI slide scanner. Liver cryosections were stained with Oil Red O as described previously (82 (link)).