Histofine simple stain kit

After receiving an intraperitoneal injection of a lethal dose of pentobarbital (Sigma-Aldrich, St. Louis, MO, USA), mice (n = 5) were transcardially perfused with cold PBS, and followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Frozen sections with 16-μm thickness were processed for the IHC with the tyramide signal amplification (TSA) technique in a free-floating manner, as in our previous reports (Okita et al., 2012 (link); Morigaki and Goto, 2015 (link)). Briefly, rabbit polyclonal antibody against TH (1:100,000) was used as a primary antibody. To detect the bound antibody, we used the Histofine Simple Stain Kit (Nichirei, Tokyo, Japan) and the TSA Plus Cyanine 3 System (Perkin Elmer, Shelton, CT, USA).

CD163 has been used as a specific marker to identify M2 macrophages [13 , 14 (link)]. Surgically resected specimens were retrieved to perform immunohistochemistry. Sections 4 μm in thickness were deparaffined and rehydrated. The sections were then subjected to endogenous peroxidase blocking in 1% H₂O₂ solution in methanol for 15 min. Antigen retrieval was performed by autoclaving the sections at 105 °C for 10 min in Dako Target Retrieval Solution (Dako, Glostrup, Denmark). Serum blocking was performed with 10% normal rabbit serum for 10 min. After H₂O₂ and serum blocking, the slides were incubated with primary mouse monoclonal anti-CD163 antibody (1:200 dilution; Leica Biosystems, Newcastle Upon Tyne, UK) at room temperature for 1 h. The secondary antibody was biotin-labeled rabbit anti-mouse IgG (1:500; Nichirei, Tokyo, Japan). Detection was performed with a DAB kit (Histofine simple stain kit; Nichirei). The sections were counterstained with hematoxylin.

Hearts were dissected free from the surrounding connective tissue, and fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into slices using a microtome (Leica RM 2235 or Leica CM1850UV; Leica, Solms, Germany). The slices were then mounted onto glass slides, and histological examinations were performed. Immunohistochemistry was performed using Histofine Simple Stain kit (Nichirei, Tokyo, Japan), according to the manufacturer’s instructions. Briefly, sections were deparaffinised with xylene and then rehydrated in a descending ethanol series. Sections were treated with 3% H₂O₂ in methanol for 15 min to inactivate endogenous peroxidases and then incubated with a primary antibody against p62 (rabbit anti-p62 antibody, 1:200; Proteintech); LC3 (rabbit anti-LC3 antibody, 1:200; Proteintech); CD68 (rabbit anti-CD68 antibody, 1:250; Abcam) at room temperature for 1 h. All sections were examined under an Olympus BX40 upright light microscope (Olympus, Tokyo, Japan).

Mice (Nihon SLC Co.; n = 5) were injected intraperitoneally with a lethal dose of pentobarbital (Sigma-Aldrich, St. Louis, MO, USA), and were then transcardially perfused with 0.01 M phosphate-buffered saline (PBS) at pH 7.2, followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.2. The brains were removed, post-fixed overnight in the same fixative at 4°C, and stored in a 10–30% sucrose gradient in 0.1 M PB at 4°C for cryoprotection. Sections were cut on a cryostat at 16-μm thickness, and stored in PBS containing 0.05% NaN₃ until use. Immunostaining was performed on free-floating sections using the tyramide signal amplification (TSA) method, according to our previous report (Okita et al., 2012 (link)). After blocking endogenous peroxidase activity, the sections were incubated in PBS containing 3% BSA for 60 min. They were then incubated in PBS-BSA with anti-PSD-95 antibody (1:10,000; Cell Signaling) for 18 h. The bound antibody was detected using the Histofine Simple Stain Kit (Nichirei, Tokyo, Japan) and the TSA-system with Cyanine3 (Perkin Elmer, Shelton, CT, USA).

Formaldehyde-fixed, paraffin-embedded tumor tissues were stained with hematoxylin and eosin (H&E) using standard techniques. Immunohistochemistry was performed using primary antibodies in conjunction with the Histofine Simple Stain kit (Nichirei, Tokyo, Japan). The following primary antibodies were used: Anti-FLAG M2 (Sigma, Burlington, MA, USA), anti-AE1/AE3 (DAKO, Santa Clara, CA, USA), anti-BCL2 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-TLE1 (Santa Cruz Biotechnology), and anti-CD163 (Bioss Antibodies, Woburn, MA, USA).