Histofine simple stain max po multi

Excised tumors were fixed in 10% formaldehyde solution overnight and were embedded in a paraffin block. From the paraffin-embedded tissues of both HER2-positive tumors (MDA-MB361) and HER2-negative tumors (MCF7), serial sections of 3 μm in thickness were prepared. After deparaffinization, the sections were incubated with Immunosaver (Nissin EM, Tokyo, Japan) at 98 °C for 45 min to retrieve the antigen and then treated with ethanol containing 0.3% H₂O₂ to block endogenous peroxidase. The sections were incubated with 5% normal goat serum (Agilent Technologies Inc., Santa Clara, CA, USA) for 10 min at room temperature and then incubated with the primary antibody against HER2 (HER2/ErbB2, clone D8F12, 1:400, Cell Signaling Technology, Danvers, MA, USA) for 60 min at room temperature. Then, the sections were treated with secondary antibodies (Histofine Simple Stain MAXPO MULTI; Nichirei Bioscience Inc., Tokyo, Japan) for 30 min at room temperature. The color was developed using 3,3′-diaminobenzidine·4HCl (DAB Substrate Kit; Nichirei Bioscience Inc.). After nuclear staining with hematoxylin, the slides were observed under a light microscope (BX53, Olympus, Tokyo, Japan).
All animals were treated in accordance with the Ethical Guidelines for Investigations of Experimental Animals of the Nippon Dental University School of Life Dentistry at Niigata (No. 219).

The immunohistochemical expression of LAG-3 was analyzed using a tissue microarray (KIN-2, AZUMAYA, Tokyo, Japan). Tissue microarrays consisted of paraffin-embedded tissue blocks constructed by extracting 3-mm cylindrical tissue cores, with appropriate histological findings. All blocks were sliced into Sect. (4 μm thick). After deparaffinization and heat-induced antigen retrieval using 1 mM ethylenediaminetetraacetic acid (pH 9.0), all sections were incubated with 3% hydrogen peroxidase for 10 min to quench the endogenous peroxidase activity. These sections were further incubated with DAKO Blocking Reagent (Protein Block Serum-Free Ready-to-use [Code X0909], DAKO North America Inc., California, USA) at room temperature for 20 min to block nonspecific antigens. LAG-3 antibody (Ab180187, clone EPR4392, diluted 1:1,500; Abcam) was applied as the primary antibody at 4 °C overnight. Secondary antibodies (HISTOFINE Simple Stain MAX-PO MULTI [NICHIREI Code 424,151], Nichirei Biosciences Inc., Tokyo, Japan) were applied at room temperature for 45 min. The antigen/antibody complex formation was performed in 3,3ʹ-diaminobenzidine tetrahydrochloride and hydrogen peroxidase substrate solution for 10 min and counterstained with hematoxylin for 5 s.