Histofine dab substrate kit

After blocking endogenous peroxidase and non-specific binding, tissue sections were stained with the primary antibodies listed in supplementary Table 2. For enzyme immunohistochemistry, we diluted the primary antibodies with R.T.U. Animal-Free Block and Diluent (Vector Laboratories, Burlingame, USA, SP5035). After incubation with Envision+System-HRP Labelled Polymer anti-rabbit (Dako, K4001) and Envision+System-HRP Labelled Polymer anti-mouse (Dako, K4003) for 30 min, tissue sections were stained with Histofine DAB Substrate Kit (Nichirei Biosciences, 425011) under a light microscope. Then, all slides were counterstained with hematoxylin, dehydrated with a series of graded ethanol solutions, and stabilized with mounting medium.

For immunohistochemistry, sections were pretreated with peroxidase-blocking solution (Dako, Glostrup, Denmark) for 5 min at room temperature to quench endogenous peroxidase. After several rinses in 1× phosphate-buffered saline (PBS; pH 7.4), tissue sections were incubated with 10 mM citrate buffer, pH 6.0, at 95°C for 20 min for antigen retrieval. After several rinses in PBS, the sections were blocked with 5% horse serum in PBS for 30 min at room temperature to eliminate non-specific antibody binding. The sections were then incubated at room temperature with rabbit anti-5-HT antibody (Nichirei Biosciences Inc., Tokyo, Japan; ready-to-use) for 60 min, monoclonal anti-human 5-HT antibody (Dako; 1:50 dilution) for 30 min, monoclonal anti-human chromogranin A (CGA) antibody (Dako; 1:100 dilution) for 30 min, or rabbit anti-human gastrin antibody (Dako; ready-to-use) for 20 min. After several rinses in PBS, the sections were incubated with the second antibody: goat anti-mouse or anti-rabbit immunoglobulins conjugated to horseradish-peroxidase-labeled polymer (EnVision™ + Dual Link System-HRP; Dako) for 30 min at room temperature. After several rinses in PBS, immunodetection was carried out using Histofine DAB Substrate Kit (Nichirei Biosciences) as the chromogen. Sections were then counterstained using Mayer’s hematoxylin solution (Wako Pure Chemical Industries Ltd., Osaka, Japan).

After deparaffinization, tissue sections were immersed in 1% H₂O₂ for 30 minutes to block endogenous peroxidase activity. The cells were washed with 0.01 M phosphate-buffered saline (PBS). To activate the target antigens, sections for anti-adiponectin (ADN) and anti-GC receptor (GCR) immunohistochemical stains were subjected to microwave treatment at 500 W power for 15 minutes. Then, the sections were cooled to room temperature and washed with PBS. Subsequently, the sections were treated with 10% normal goat serum (Nichirei Bioscience, Tokyo, Japan) and anti-GCR immunohistochemical stain sections were treated with 0.01% normal goat serum (Nichirei Bioscience) for 30 minutes. Then, anti-ADN immunohistochemical stain sections were treated with anti-rabbit ADN polyclonal antibody (1:100) (ab47852; Abcam, Cambridge, UK), and anti-GCR immunohistochemical stain sections were incubated with GCR alpha antibody (1:50) (ab3580; Abcam) overnight at room temperature. Sections were washed with PBS and, then, incubated with anti-rabbit immunoglobulin peroxidase-conjugated antibody (Simple Stain Rat MAX-PO [MULTI]; Nichirei Bioscience) for 1 hour at room temperature. The sections were subsequently washed with PBS again, stained with 3,3'-diaminobenzidine chromogen solution (Histofine DAB Substrate Kit; Nichirei Bioscience), and counterstained with hematoxylin.

Hepatocellular carcinoma cells of 5.0 × 10⁴ per well in a six‐well plate were fixed with 4% paraformaldehyde. Subsequently, Cells were treated with serum‐free ready‐to‐use protein block (Dako) and then incubated overnight with rabbit anti‐SUV420H1 polyclonal antibody (LS‐C359286; LifeSpan BioSciences) at 1:200 dilution ratio at 4°C. Cells were stained with anti‐rabbit EnVision HRP (Dako) and Histofine DAB substrate kit (Nichirei). The experiment was done in triplicate.