Histofine simple stain max po g

The medium was washed three times with PBS to thoroughly remove the medium from the spheroids. The spheroids were fixed with neutral buffered 10% formalin for 10 min and embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E) staining and immunohistochemical staining for vimentin, ALB, and von Willebrand Factor (vWF). Blocking was carried out by adding PBS containing 5% skim milk, and immunohistochemistry was carried out by incubation overnight at 4 °C with polyclonal goat anti-dog albumin antibody (Bethyl Laboratories Inc., TX, USA), monoclonal mouse anti-vimentin antibody (clone V9; Agilent Technologies, California, USA), or polyclonal rabbit anti-human vWF antibody, HRP conjugated (Agilent Technologies). The corresponding secondary antibodies for albumin and vimentin included anti-goat antibody (Histofine Simple Stain MAX-PO(G), NICHIREI BIOSCIENCE, Tokyo, Japan) and anti-mouse antibody (Histofine Simple Stain MAX-PO(M), NICHIREI) for 30 min. As a negative control, 2 μg/mL normal goat IgG (R&D Systems, Minnesota, USA) and 1 μg/mL normal mouse IgG (Merck KGaA, Darmstadt, Germany) was used. All antibodies were diluted using an antibody diluent with background reducing components (Agilent Technologies). As color reaction, histofine SAB-PO(M) kit (NICHIREI) containing DAB (3,3′-diaminobenzidine tetrahydrochloride) and H₂O₂ was used.