TMA construction by using individual FFPE samples was performed as previously described (8 (link)). Each tissue array block contained up to 50 specimens, which allowed a total of 394 specimens to be mounted on 20 blocks. Serial sections (4 µm) of the FFPE blocks were analyzed for protein expression with immunohistochemistry. A pathologist performed a test procedure by using a control human TMA panel (19 (link)) according to the manufacturer’s information and evaluated the quality of positive and negative controls in immunohistochemistry. Antibodies against RIPK3, MLKL, and PELI1 (F-7) were used due to their essential roles in the necrosome formation and modulation (3 (link)). In addition, antibodies against P53, γH2AX, ATM, Chk2pT68, BRCA1pS1423, and ERCC1 were used due to their well-defined roles in cellular responses to DNA damage (1 (link),8 (link),9 (link)). Information for the antibodies used in this study is summarized in Table S2 . All immunohistochemical staining was performed with a BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ) and the i-View detection kit (Ventana Medical System) by following the standard operating procedure at SuperBioChips Laboratory, Seoul, South Korea.
I view detection kit
Manufactured by Roche
Sourced in United States
The I-View detection kit is a laboratory equipment product designed for use in scientific research and analysis. It serves as a tool for detecting and analyzing specific targets or analytes within a sample. The core function of the I-View detection kit is to provide accurate and reliable data to support scientific investigations, without interpretation or extrapolation beyond its intended purpose.
Automatically generated - may contain errors
Lab products found in correlation
Benchmark xt autostainer, by Roche (2 mentions)
Ventana benchmark xt, by Roche (2 mentions)
Ventana automated immunostainer, by Roche (1 mentions)
Ez prep, by Roche (1 mentions)
Confirm anti ki 67 30 9, by Roche (1 mentions)
Discovery xt, by Roche (1 mentions)
Benchmark ultra automated immunostainer, by Roche (1 mentions)
Bond max, by Leica (1 mentions)
Anti rabbit secondary antibody, by Merck Group (1 mentions)
Benchmark autostainer, by Roche (1 mentions)
Cd133, by Proteintech (1 mentions)
Cd147, by Cell Signaling Technology (1 mentions)
9 protocols using i view detection kit
1
Tissue Microarray Protocol for Protein Expression
2
ALK Immunohistochemistry Protocol
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ALK IHC was carried out on 3-μm thick tissue using a Ventana automated immunostainer (Ventana Medical Systems, Tucson, AZ) according to the manufacturer's protocol. Briefly, the slides were deparaffinized using EZ Prep (Ventana Medical Systems) at 75°C for 4 minutes and heat pretreatment at 100°C for 20 minutes. The antibody for ALK (mouse monoclonal, clone 5A4, Novocastra, Newcastle, United Kingdom) was diluted to 1:30 and incubated at 42°C for 2 hours. Signals were detected with an iView detection kit (Ventana Medical Systems) based on a streptavidin-biotin method. Mayer's hematoxylin was used for counterstaining for 2 minutes at room temperature. ALK expression was semiquantitatively assessed based on the intensity of staining and the proportion of stained cells and scored by two independent pathologists (IG Do and KM Kim). An IHC score was assigned as 0 (no staining), 1+ (faint cytoplasmic staining in ≤10% of tumor cells), 2+ (moderate, smooth cytoplasmic staining), and 3+ (intense, granular cytoplasmic staining). IHC scores of 2+ or 3+ were regarded as ALK IHC positive.
3
Immunohistochemical Ki-67 Analysis of FTs
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Immunohistochemical Ki‐67 staining of 5 representative FTs was performed in sections from paraffin‐embedded tissue blocks with the avidin‐biotin‐peroxidase technique. Five micrometer‐thick sections were cut from the tissue specimens and placed on poly‐l ‐lysine‐coated glass slides. Sections were deparaffinized with xylene and rehydrated in graded alcohol. Endogenous peroxidase was blocked by immersing the sections in 0.1% hydrogen peroxidase in absolute methanol for 20 min. For antigen retrieval, tissue sections were heated in a pressure cooker in 10 mM citric acid monohydrate, pH 6.0 for 5 min, and then incubated with the primary antibody at room temperature. The primary antibody used was CONFIRM anti‐Ki‐67 (30–9) (Ventana Medical Systems, Tucson, AZ). Immunohistochemistry was performed with the Ventana BenchMark XT slide processing system and the iView detection kit (Ventana Medical Systems, Tucson, AZ). All slides were counterstained with hematoxylin, dehydrated, and mounted. Negative controls were performed by omitting the primary antibody.
4
MYOD1 IHC on FFPE Sections
5
Immunohistochemical Evaluation of p16 Expression
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After deparaffinisation and rehydration, heat-induced antigen retrieval was performed by microwave oven for 20 min in a citrate buffer. Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in methanol for 10 min, and then nonspecific antibody binding was blocked by 10% normal goat serum. IHC was performed using the Benchmark Ultra automated immunostainer (Ventana Medical Systems, AZ, USA). Immunoreactivity against anti-p16 mouse monoclonal antibody (clone JC8, Santa Cruz Biotechnology, Inc., CA, USA) was visualised using the iVIEW detection kit (Ventana Medical Systems) containing the biotin-conjugated secondary antibody, streptavidin-hydrogen peroxide conjugate, and diaminobenzidine. The p16 IHC was interpreted as ‘positive’ when the percentage of stained nuclear and cytoplasmic cells was > 70% of cancer cells. Patchy or focal expression in the surface epithelial cells was regarded as negative.
6
Tissue Microarray Analysis of DNA Damage Proteins
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TMA construction from individual FFPE samples was performed as previously described [8] . Each tissue array block contained up to 50 specimens, which allowed all 394 specimens to be contained in 20 blocks.
Commercially available antibodies were chosen for RIPK3, MLKL (EPR17514), and PELI1 (F-7); these proteins were chosen as they are known to play essential roles in necrosome formation and modulation (Supplementary Table 1) [3] . P53 (DO-7), gH2AX (JBW301), ATM (7C10D8), Chk2pT68, BRCA1pS1423, and ERCC1 (8F1) were chosen because of their well-de ned roles in cellular responses to DNA damage [1, 8, 9] .
Serial sections (4 mm) from the FFPE blocks were analyzed for protein expression by immunohistochemistry. All antibodies were tested using a human control TMA panel [21] . A pathologist performed the test procedure with manufacture's information and evaluated the quality of positive and negative controls in immunohistochemistry.
All immunohistochemical staining was performed with a BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ) and i-View detection kit (Ventana Medical System) by following a standard operating procedure at SuperBioChips Laboratory, Seoul, South Korea.
Commercially available antibodies were chosen for RIPK3, MLKL (EPR17514), and PELI1 (F-7); these proteins were chosen as they are known to play essential roles in necrosome formation and modulation (Supplementary Table 1) [3] . P53 (DO-7), gH2AX (JBW301), ATM (7C10D8), Chk2pT68, BRCA1pS1423, and ERCC1 (8F1) were chosen because of their well-de ned roles in cellular responses to DNA damage [1, 8, 9] .
Serial sections (4 mm) from the FFPE blocks were analyzed for protein expression by immunohistochemistry. All antibodies were tested using a human control TMA panel [21] . A pathologist performed the test procedure with manufacture's information and evaluated the quality of positive and negative controls in immunohistochemistry.
All immunohistochemical staining was performed with a BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ) and i-View detection kit (Ventana Medical System) by following a standard operating procedure at SuperBioChips Laboratory, Seoul, South Korea.
7
Immunohistochemical Analysis of Tumor Samples
8
Immunostaining of CD133 and CD147 in Tissue Sections
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Serial 4 μm sections were applied to 3-aminopropyltriethoxysialne-coated slides (Sigma), deparaffinized, and rehydrated in xylene and serially diluted ethanol. Endogenous peroxidase was blocked by incubation in a 3% aqueous hydrogen peroxide solution, after which heat-induced antigen retrieval was performed. Primary antibodies against CD133 (1:100; #66666-1) (Proteintech, Rosemont, IL, USA) and CD147 (1:200; #13287) (Cell Signaling Technology, Danvers, MA, USA) with a benchmark autostainer (Roche Tissue Diagnostics, Oro Valley, AZ, USA) were used following the manufacturer’s protocol. The primary antibody was incubated at RT for 32 min, after which the sections were labeled with an automated immunostaining system using an I-View detection kit (Roche Tissue Diagnostics, Oro Valley, AZ, USA). The immunostained sections were lightly counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene.
9
Quantifying Podocyturia via Immunocytochemistry
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Podocyturia was assessed using immunocytochemical detection of podocalyxin-positive cells on slides prepared from fresh urine samples with a filter imprint technique where the whole sample was filtered through isopore polycarbonate membrane filters with 5-µm pores (Merck Millipore, Dublin, Ireland; TMTP04700) using the Microfil System (Millipore 3-Place Manifold MIAC03P01, MIHA WGO 72 funnels; Millipore Intertech, Bedford, MA, USA). Cells collected on the filter surface were immediately transferred to a pair of glass slides by the imprint technique and fixed with M-FIX spray fixative (3981; Merck, Darmstadt, Germany). Immunocytochemical staining with monoclonal antibody against podocalyxin (PHM5, Millipore MAB430; Millipore, Bayswater, VIC, Australia) was performed using the automated immunostaining system Ventana Benchmark XT (Roche Diagnostics, Rotkreuz, Switzerland) and the iView detection kit (Roche Diagnostics). The number of podocalyxin-positive cells per slide was counted by an experienced cytologist using a light microscope at magnification 200×. Results were expressed as the number of podocytes per 100 mL of urine (UPodo/100 mL) and per gram of Cr in urine (UPodo/g Cr).
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