Bond polymer refine detection kit

Slides were covered with covertiles (Bond Universal Covertiles, Leica biosystems) and baked for 10 mins at 57°C. Slides were deparaffinizied in dewax solution and rehydrated in decreasing concentrations of EtOH. Tissue sections were then incubated in Ag retrieval solution (pH 6 or 9) at 95°C for 20 mins. Tissue sections were incubated in 3% hydrogen peroxide (Bond Polymer Refine Detection Kit DS9800, Leica Biosystems) for 15 mins to block endogenous peroxidases. Next, tissue sections were incubated in serum-free protein block solution (Dako X0909) for 30 mins to block nonspecific antibody binding. After the first staining cycle, Fab fragments (AffiniPure Fab Fragment Donkey anti-mouse (715–007-003) or anti-rabbit IgG (711–007-003)) against that primary antibody species were used to block carryover staining whenever there was a repeat of same primary antibody species. Primary antibody staining was performed for 1 hour at room temperature followed by secondary antibody staining. Polymer detection system (Bond Polymer Refine Detection Kit #DS9800, Leica Biosystems) was used for horseradish peroxidase signal amplification. Chromogenic revelation was performed using ImmPact AEC (3-amiino99-ethylcarbazole) substrate (Vector Laboratories, SK4205) for preset incubation times. Slides were counterstained with hematoxylin (Bond Polymer Refine Detection Kit, DS9800, Leica Biosystems.

Immunohistochemistry was carried out with antibodies directed against PD-L1 [dilution 1:100, E1L3N, Cell Signaling, Danvers, USA (catalog #13684)], PD-1 [dilution 1:100; clone MRQ-22, Cell Marque, Rocklin, USA (#315M-96)], and VISTA [1:500; clone D1L2G, Cell Signaling (#64953)]. Immunostaining of PD-L1 and PD-1 was performed with the autostainer Bond™ Max System (Leica Microsystems GmbH, Wetzlar, Germany). The immunoreaction was visualized with the Bond™ Polymer Refine Detection Kit [brown labeling; Novocastra; Leica Microsystems GmbH, Wetzlar, Germany (#DS9800)]. Immunostaining of VISTA was performed manually: Following antigen retrieval in citrate buffer (pH6), specimens were incubated with hydrogen peroxide block and Ultra V Block [both Thermo Scientific, Braunschweig, Germany (TA-125-HP and TP-125-HL)] to avoid unspecific reactions. The immunoreaction was visualized with the ImmPRESS-HRP-Universal–Antibody Polymer and the NovaRED substrate kit [both VectorLabs, Peterborough, United Kingdom (#SK-4800)]. Counterstaining was carried out with hematoxylin [Dr. K. Hollborn & Söhne GmbH & Co KG; Leipzig, Germany (#88663)].
Germinal centers of lymph follicles served as internal positive control for PD-L1 and PD-1.

Tissue specimens from clinical GC samples and xenograft tumors derived from GC cells were fixed with 10% neutralbuffered formalin, and 4-μm paraffin sections were prepared. After rehydration, sections were stained with hematoxylin and eosin for histologic assessment, or were immunostained after antigen retrieval using a Bond-max automated immunostainer (Leica Microsystems, Newcastle upon Tyne, UK). The primary antibodies used were against FOXO1 (1:40, C29H4, Cell Signaling Technology), phospho-FOXO1^Ser256 (pFOXO1; 1:60, Cell Signaling Technology), CD31 (1:100, M20, Santa Cruz Biotechnology), HIF-1α (1:50, provided by Dr. Jong-Wan Park at Seoul National University), VEGF (1:200, C1, Santa Cruz Biotechnology), and SIRT1 (1:100, H300, Santa Cruz Biotechnology). Antibody binding was detected with the Bond Polymer Refine Detection Kit (Leica Microsystems). All immunostained sections were lightly counterstained with Mayer’s hematoxylin. Throughout the above analysis, negative controls were prepared by omitting the primary antibody. The results of immunostaining were evaluated by two pathologists (Y.K. and J.-S.P.), who were blinded to the origin of the samples. For statistical analysis, the results of immunostaining for proteins were considered positive if immunoreactivity was seen in ≥ 10% (cytoplasmic pFOXO1 and nuclear SIRT1) or ≥ 5% (nuclear HIF-1α) of the neoplastic cells.

Tissue specimens from clinical gastric carcinoma samples and xenograft tumours derived from GC cells were fixed with 10% neutral-buffered formalin and 4-μm paraffin sections were then prepared. After rehydration, sections were stained with haematoxylin and eosin for histologic assessment, or were immunostained after antigen retrieval using a Bond-max automated immunostainer (Leica Microsystems, Newcastle, UK). The primary antibodies used were against HER2 (1 : 100), pFOXO1 (1 : 60) and FOXO1 (1 : 40). Antibody binding was detected with the Bond Polymer Refine Detection kit (Leica Microsystems). All immunostained sections were then lightly counterstained with Mayer's haematoxylin. Throughout the above analysis, negative controls were prepared by omitting the primary antibody.
For tissue array analysis of human GC specimens, HER2 immunostaining in cancer cells was scored in accordance with the HER2 scoring system for GC as described in a previous study (Kim et al, 2011 (link)). Briefly, cases showing weak to strong staining of the entire or basolateral membrane in ⩾10% of the tumour cells were considered HER2 immuno-positive. For the analysis of FOXO1 staining, cases showing cytoplasmic expression of pFOXO1 (inactive form of FOXO1) in ⩾10% of the tumour cells were considered pFOXO1 immuno-positive (Kim et al, 2007 (link)).

Formalin-fixed paraffin-embedded myometrial (n = 5 per group), cervical (n = 5–7 per group), and chorioamniotic membrane (n = 5 per group) tissues were cut in five-μm-thick sections. Slides were deparaffinized in xylene and hydrated with decreasing concentrations of ethanol. Immunohistochemistry staining for IFNe (mouse anti-human IFNe, Clone 983338; R&D Systems) was performed using the Leica Bond Max automatic staining system (Leica Microsystems; Wetzlar, Germany). The Bond™ Polymer Refine Detection Kit (Leica Microsystems) was used to detect the chromogenic reaction of horseradish peroxidase upon oxidation of 3’3-Diaminobenzidine (DAB). Mouse IgG2B isotype (Cat. No. MAB004; R&D Systems) was used as the negative control. Brightfield images were taken using the Vectra Polaris Multispectral Imaging System and inForm software version 2.5.1. Representative images were taken at 200X magnification.