Novolink max polymer detection system

Immunohistochemical staining for LPP3 was undertaken using mouse monoclonal antibody clone 7H7D3 (Biosensis, Temecula, CA), which has previously been validated, including for immunohistochemistry [95 (link)]. Formalin-fixed paraffin-embedded tissue samples of non-atherosclerotic aorta (n = 5), and atherosclerotic aorta (n = 5) obtained with full ethical approval from the National Research and Ethics Service (Oxfordshire Research and Ethics Committee A: reference 04/Q1604/21), were immunostained using the Bond Max™ fully automated immunohistochemistry system (Leica Microsystems, Wetzlar, Germany). Heat induced antigen retrieval was performed using the EDTA-based Novocastra Bond Epitope Retrieval Solution 2 (pH 9.0) for 30 minutes and the NovolinkTM max polymer detection system (Leica Microsystems) was used, as per the manufacturer’s instructions. Detection reagents only were used as a negative control. Slides were mounted in Aquatex mounting medium (Merck, Darmstadt, Germany). Stained sections were photographed with a Nikon DS-FI1 camera with a Nikon DS-L2 control unit (Nikon, Kingston-upon-Thames, UK) and an Olympus BX40 microscope (Olympus, Watford, UK). All immunohistochemical staining was analyzed by and performed under the supervision of an experienced board certified consultant pathologist (ES).

According to the individual case and aims, immunohistochemistry (IHC) was performed. A NovolinkTM Max-Polymer detection system (Novocastra, Newcastle, UK) was used according to the manufacturer’s instructions. Slides were incubated with a panel of specific antibodies, for which detailed characteristics are described in Table 2.

For the immunohistochemical study, sections were deparaffinised, hydrated and antigen retrieval was performed in a pressure cooker in 10 mmol/L sodium citrate buffer, pH 6.0, for 2 minutes (min). Slides were cooled for 10 min at room temperature and rinsed twice in triphosphate buffered saline (TBS) for 5 min. The NovolinkTM Max-Polymer detection system (Novocastra, Newcastle, UK) was used for visualisation, according to the manufacturer’s instructions. After blocking endogenous peroxidase with 3% hydrogen peroxide in methanol for 10 min, sections were incubated overnight at 4°C, with a polyclonal antiserum against H. pylori (RBK012; Zytomed, German) which shows immunoreactivity with a wide range of bacteria belonging to the Helicobacter genus. Sections were rinsed with TBS between each step of the procedure. Colour was developed for up to 7 min at room temperature with 3,3′-diamino-benzidine (DAB) (Sigma, St. Louis, MO) and sections were then lightly counterstained with haematoxylin, dehydrated and mounted. Positive immunoreactivity was recorded as a distinct golden-brown labelling of the bacteria located on mucosal surface, in gastric pits or glands and in parietal cells.

Sections were deparaffinized in xylene and rehydrated in sequential graded alcohols, and antigen retrieval was accomplished in a water bath in a 10 mmol/L sodium citrate buffer at pH 6.0 for 20 minutes (min). Slides were chilled for 10 min at room temperature and washed twice for 5 min in triphosphate buffered saline (TBS). For visualization, the NovolinkTM Max-Polymer detection system (Novocastra^®, Newcastle, UK) was used, following the manufacturer’s instructions. Sections were incubated overnight at 4 °C, with a polyclonal anti-serum against H. pylori (RBK012; Zytomed, Berlin, German) (1:200) that has been shown to have immunoreactivity with a wide assortment of Helicobacter genus bacteria [4 (link)]. Sections were rinsed with TBS between each step of the procedure. Color was developed with 3,3′-diamino-benzidine (DAB) (Sigma^®, St. Louis, MO, USA), and sections were then lightly counterstained with hematoxylin, dehydrated, and mounted. Negative controls were performed by replacing the primary antibody with an antibody of the same immunoglobulin isotype at the same original concentration. Positive immunoreactivity was recorded as a distinct golden-brown labelling of the bacteria located on the mucosal surface, in gastric pits or glands, and inside parietal cells.

SIRT1 and SIRT7 protein expression were studied by IHC. Sections (3μm thick) from the FFPE samples, mounted on glass slides, were deparaffinised in xylene and hydrated through a graded alcohol series. Antigen retrieval was accomplished by microwaving the slides in EDTA buffer (20′ and 40′ for SIRT1 and SIRT7, respectively) and endogenous peroxidase activity was blocked with 0.6% hydrogen peroxide.
Protein detection was performed using the NovolinkTM Max Polymer Detection System (Leica Biosystems, Nussloch, Germany), according to manufacturer instructions. Slides were incubated overnight at 4°C with mouse monoclonal antibodies specific for SIRT1 (#ab32441, 1:750, Abcam, Cambrige, United Kingdom) and SIRT7 (SC-365344, 1:100, Santa Cruz Biotechnology, Dallas, USA). The slides were washed, developed with diaminobenzidine chromogen and counterstained with hematoxylin. Finally, after dehydration and diaphanization, slides were mounted in Entellan^® (Merck-Millipore, Germany). Colorectal mucosa and kidney parenchyma sections were used as positive controls for SIRT1 and SIRT7, respectively.
Semi-quantitative assessment of SIRT1 and SIRT7 nuclear protein expression was done using Allred score [32 (link), 33 (link)], by estimating the proportion and intensity of positive cells (range 0, 2 to 8). Scores of 3 or greater were defined as positive.

Cross-sections of 4.0 μm thickness were also used for immunohistochemistry. The samples were incubated with the primary antibodies, anti-RANKL (sc-377079, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-OPG (sc-390518, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), to evaluate bone resorption and bone remodeling. The secondary antibodies and antibody detection reaction (NovolinkTM Max Polymer Detection System) were performed according to the manufacturer’s recommended protocol (Leica Biosystems, Buffalo Grove, IL, USA). Diaminobenzidine (DAB) was used as a substrate chromogen to reveal the specific markings, followed by hematoxylin counterstaining. Ten images of each bone sample were captured at 400× using a Leica DM2000 microscope (Leica Microsystems, Wetzlar, Germany) and quantified using ImageJ software.

Immunohistochemistry (IHC) was performed on 3-μm paraffin sections of all 44 LSCC cases. For ALCAM antigen retrieval, sections were incubated in a steam oven while submerged in a trilogy buffer solution (Cell Marque), for 30 min at 98 °C. Sections were then incubated with the primary monoclonal antibodies against ALCAM (Sigma, St. Louis, MO, USA, HPA010926, working dilution 1:1000), for at least 12 h. Formalin-Fixed Paraffin-Embedded (FFPE) prostate carcinoma samples served as positive control staining. As the negative control, the primary antibody was replaced by the diluent solution. The detection system used was the NovoLinkTM Max Polymer Detection System (Leica Biosystems, Wetzlar, Germany), following the protocol described by the manufacturer, using diaminobenzidine as substrate (Dako). Sections were counterstained with Harris’ hematoxylin. Scored cases were considered positive when at least 1% of epithelial cells were stained. LSCC samples were categorized as low and high ALCAM protein expression using the median number of positive cells as the cut-off. Samples with positive epithelial cells lower than median value were classified as low ALCAM tumors and samples with positive epithelial cells equal or higher than median value were classified as high ALCAM tumors.