The 5-µm-thick sections were deparaffinized and rehydrated using xylene (100%) and descending ethanol series (75–100%). Each incubation was performed for 10 min at room temperature. Subsequently, 0.3% H2O2 for 10 min at room temperature was used to block endogenous peroxidase activity. Subsequently, the antigens were retrieved using sodium citrate (pH 6.0, 0.01 M) at 98°C for 20 min). Non-specific binding was blocked using 5% BSA (Sangon Biotech Co., Ltd.) at room temperature for 1 h. The sections were stained with CALB1 primary antibody (Abcam) at 4°C overnight and visualized with the appropriate horseradish-conjugated secondary antibody using an EnVision system (Agilent Technologies, Inc.) according to the manufacturer's protocol. Subsequently, the slides were developed with 3,3′-diaminobenzidine at room temperature for 4 min and counterstained with hematoxylin at room temperature for 4 min. The staining intensity and the protein expression levels were evaluated using the Vectra2 system (PerkinElmer, Inc.). The staining was assessed using the H-score as previously described (20 (link)), which was calculating by multiplying the percentage of positive cells by the staining intensity of the tumor cells (ranging between 0 and 3).
Vectra 2 system
Manufactured by PerkinElmer
Sourced in United States
The Vectra 2 system is a high-performance multispectral imaging platform designed for advanced biological research. It combines automated slide scanning, spectral unmixing, and quantitative analysis to enable comprehensive visualization and assessment of tissue samples.
Automatically generated - may contain errors
8 protocols using vectra 2 system
1
CALB1 Immunohistochemical Staining Protocol
2
NSCLC Tissue Array Immunohistochemistry
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NSCLC tissue arrays were purchased from Shanghai OUTDO Biotech. The tissue arrays were deparaffinized and rehydrated prior to antigen retrieval and blocking of endogenous peroxidase activity. The sections were washed three times with 0.01 mol/L PBS (2 mmol/L NaH2PO4, 8 mmol/L Na2HPO4 and 150 mmol/L NaCl). Then, 0.01 mol/L PBS containing 5% normal goat serum and 0.1% Triton X-100 was used to block the sections, followed by incubation with an anti-PPDPF antibody (1:100; Proteintech, 19912-1-AP) overnight at 4 °C. After three washes in PBS and treatment with adjuvant for 20 min at 37 °C, the sections were incubated with a secondary antibody for 20 min at 37 °C. The immunohistochemical reaction was visualized with 3,3'-diaminobenzidine (DAB) for 2 min. All sections were counterstained with hematoxylin. Both the staining intensity and extent of protein expression were automatically scored with a Vectra 2 system (Perkin-Elmer, USA).
3
Immunohistochemical Evaluation of MTF2 Expression
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Paraffin-embedded tissue sections were deparaffinized, rehydrated, subjected to antigen retrieval, and the endogenous peroxidases were blocked. After washing the section thrice with 0.01 mol/L PBS (8 mmol/L Na2HPO4, 2 mmol/L NaH2PO4, and 150 mmol/L NaCl), the sections were blocked in 0.01 mol/L PBS containing 0.3% Triton X-100 and 5% BSA. Next, sections were incubated with anti-MTF2 (1:200) antibody (Abcam) at 4 °C overnight. The sections were washed thrice with 0.01 M PBS and incubated with secondary antibody (1:500) for 2 h. After washing with 0.01 mol/L PBS and 0.05 M Tris-HCl (pH 7.6) solution twice, sections were visualized with 0.03% 3,3′-diaminobenzidine in 0.05 M Tris-HCl (pH 7.6). The extent and staining intensity of protein were scored automatically using the Vectra 2 system (Perkin-Elmer, USA). The outcome of staining was determined using the H-score, defined by the equation: H-score = 100ΣPi × i. The staining intensity (i) of the decorated tumor cells was graded from “0 to 3”, and Pi is the percentage of the stained cells at various intensities.
4
Quantifying NET1 Expression in Paraffin-Embedded Tissues
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Paraffin-embedded tissue sections were deparaffinized and rehydrated before being subjected to endogenous peroxidase inhibition. The sections were rinsed thrice in PBS (0.01 mol/l), and blocked with PBS (0.01 mol/l) containing 5% BSA and 0.3% Triton X-100 for 1 hour. After incubation with anti-NET1 at 4°C overnight, the samples were incubated again with the secondary antibody at room temperature for 2 hours. After being developed with 3,3’-diaminobenzidine (0.03%) and H2O2 (0.003%) in Tris-HCl (0.05 mol/l, pH 7.6), the extent and staining intensity examined automatically by Vectra 2 system (PerkinElmer, USA) were calculated using H-score as previously described [18 (link)]. According to the median score of NET1, tissue samples with final H-scores of <81 and ≥81 were categorized as NET1 “low” and “high” expression, respectively.
5
Immunohistochemical Analysis of YTHDC2 in NPC
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Paraffin-embedded NPC samples were collected from Xiangya Hospital of Central South University. Tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval, and endogenous peroxidases were blocked. The sections were washed three times with 0.01 mol/L PBS (2 mmol/L NaH2PO4, 8 mmol/L Na2HPO4, and 150 mmol/L NaCl). Then, 0.01 mol/L PBS supplemented with 5% normal goat serum and 0.1% Triton X-100 was used to block the sections, followed by an incubation with anti-YTHDC2 (1:1,000; Sigma, HPA037364) overnight at 4°C. After three times washing in PBS and treatment with adjuvant for 20 min at 37°C, the sections were exposed to secondary antibody for 2 h at 37°C. The immunohistochemical reaction was visualized with 3, 3, 0-diaminobenzidine (DAB) for 3 min. All sections were counterstained with haematoxylin. Both the staining intensity and extent of protein expression were automatically scored by Vectra 2 system (Perkin-Elmer, USA).
6
Immunohistochemical Analysis of PRMT4 Protein
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Paraffin-embedded tissue sections were deparaffinized and rehydrated before being subjected to endogenous peroxidase inhibition. The sections were rinsed three times in PBS (0.01 mol/l) and blocked with PBS (0.01 mol/l) containing 5% BSA and 0.3% Triton X-100 for 1 h. After incubation with anti-PRMT4 antibody (Proteintech, #55246-1-AP, 1:200) at 4°C overnight, the samples were incubated again with the secondary antibody at room temperature for 2 h. After being developed with 3,3'-diaminobenzidine (0.03%) and H2O2 (0.003%) in Tris-HCl (0.05 mol/l, pH 7.6), the extent of staining and the staining intensity were examined automatically by the Vectra 2 system (PerkinElmer, USA) and they were calculated using the H-score as previously described 18 (link).
7
Colorectal Cancer RYR2 Expression Study
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A total of 195 patients diagnosed pathologically with colorectal cancer were enrolled in this study. Samples were collected at The First People's Hospital of Huzhou from 2018 to 2019 in compliance with the protocol for tissue collection approved by the Ethics Committee of First Affiliated Hospital, Huzhou University (approval number: 2020KYLL002). All methodologies conformed to the standards set by the Declaration of Helsinki. Informed consent was signed by all patients and all experiments were approved by the Ethical Committee. Detailed pathological examination parameters were recorded and available for analysis. Tissue microarray (TMA) was constructed from CRC samples embedded in paraffin, and stained with anti‐RYR2 (diluted 1 : 100), followed by scanning, photographing and scoring using the Vectra2 system (PerkinElmer, Waltham, MA, USA).
8
Immunohistochemical Analysis of Protein Expression
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Paraffin-embedded tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Subsequently, the endogenous peroxidase activity of sections was blocked with 3% H 2 O 2 in methanol. After washing with 0.01 M PBS (15 mM NaCl, 8 mM Na 2 HPO 4 , and 2 mM NaH 2 PO 4 ) three times, the sections were blocked by 0.01 M PBS supplemented with 5% normal goat serum and 0.3% Triton X-100. Next, sections were incubated with primary antibodies against XAF1 (1:100; Affinity), RNF114 (1:100; Absci), and JUP (1:50; Cell Signaling Technology), respectively, overnight at 4°C. After washing with 0.01 M PBS three times, the sections were incubated with a secondary antibody (1:500) for 2 h. After washing once with 0.01 M PBS and twice with 0.05 M Tris-HCl (pH = 7.6) solution, the sections were visualized with 0.03% 3,3′-diaminobenzidine in 0.05 M Tris-HCl (pH 7.6) and counterstained with hematoxylin. Images were captured at room temperature using Echo Revolve Hybrid Microscope (Echo) using 20 × objective at bright field mode. The extent and staining intensity of protein were scored automatically by Vectra 2 system (PerkinElmer).
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