N histofine simple stain rat max po

To gain insight into the expression of Bag-1 in the IVD, freshly isolated spines from 11-week-old rats were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), decalcified and embedded in paraffin wax. Sagittal sections were deparaffinized in xylene, rehydrated through a graded ethanol series and incubated with antibodies against Bag-1 (#ab32109; Abcam plc, Cambridge, UK) in 2% normal goat serum in tris buffered saline with Tween 20 (TBS-T) at a 1:50 dilution overnight at 4 °C. The sections were washed thoroughly and then stained with N-Histofine^® Simple Stain™ Rat MAX PO (NICHIREI BIOSCIENCES INC. Tokyo, Japan). Nuclei were stained with haematoxylin. The color was developed using 3, 30-diaminobenzidine (Vectastain Universal Quick Kit; Vector Laboratories Inc., Newark, CA, USA) and the slides were examined under a microscope (BX63; Olympus, Tokyo, Japan).

The implanted SF graft was hollowed out together with the skin with surgical scissors and divided in half. In addition, the central part of the graft was cut 4 mm transversely, and the sutured part of the remaining native blood vessel and artificial vascular graft was cut longitudinally. These samples were fixed with ethanol for histological analyses. The fixed samples were embedded in paraffin and processed for hematoxylin and eosin (H&E) and Masson’s trichrome (MTC) staining. Sections for immunohistochemical staining were incubated with primary antibodies, including α-smooth muscle actin (α-SMA; clone 1A4; Sigma-Aldrich, Inc., Tokyo, Japan) and CD31 anti-rat antibody (BD Biosciences, Inc., Tokyo, Japan). The A-SMA sample was incubated with biotinylated anti-mouse immunoglobulin secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA), and subsequent color development was obtained using Vectastain ABC-AP (Vector Laboratories, Inc., Burlingame, CA, USA). The CD31 sample was incubated with N-Histofine Simple Stain Rat MAX PO (Nichirei Biosciences, Inc., Tokyo, Japan), and subsequent color development was obtained using Vectastain ABC-AP (Vector Laboratories, Inc., Burlingame, CA, USA).