Paraffin-embedded tumor tissues were consecutively cut into 5 µm thick sections and processed for immunohistochemical staining using the endothelial cell marker CD31, as described previously (23 (link)). Briefly, Antigen retrieval was carried out using the EnVisionTM FLEX Target Retrieval Solution (pH =9.0, Dako), followed by incubation with a rabbit polyclonal CD31 antibody (dilution: 1:300, Proteintech, Rosemont, USA) at 4 °C overnight. Sections were then incubated with the secondary antibody (PV-9000, Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) for 1 hour, followed by color development with 3,3’-diaminobenzamine in Tris-HCl (50 mmol/L, pH =7.5) containing 0.005% hydrogen peroxide, and counterstaining with hematoxylin.
Pv 9000
Manufactured by Zhongshan Jinqiao Biotechnology
Sourced in China
The PV-9000 is a versatile, high-performance laboratory instrument designed for advanced analytical applications. It features precise temperature control, reliable performance, and user-friendly operation. The PV-9000 is suitable for a wide range of scientific and research purposes, providing consistent and accurate results.
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5 protocols using pv 9000
1
Immunohistochemical Staining of Endothelial Cells
2
Evaluating MCT1 and MCT4 Expression in Lung Tumors
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IHC staining was performed to evaluated the expression of MCT1 and MCT4, in both tumors and paired adjacent lung tissues in each case. In brief, the formalin-fixed paraffin-embedded tissues were cut into a series of 5 µm-thick sections. The sections were de-paraffinized, rehydrated and underwent antigen retrieval using Dako EnVisionTM FLEX Target Retrieval Solution (pH 9.0) at 95 °C for 20 minutes. Anti-SLC16A3 (MCT4) antibody (Rabbit polyclonal antibody, HPA021451, Sigma-Aldrich), and anti-MCT1 antibody (Rabbit polyclonal antibody, ab238825, Abcam) were used as primary antibodies, and were diluted at a ratio of 1:100. After 2 hours of primary antibody incubation at room temperature, incubation with a secondary antibody (PV-9000, Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) was performed at room temperature for 30 minutes. Finally, 3,3’-diaminobenzamine and hematoxylin were used for coloration of the immune complex and nucleus, respectively. The expression of MCT4 and MCT1 was assessed by multiplying the staining intensity score and the percentage score as described in our previous study (26 ). A final score ≥6 was defined as high expression.
3
Apelin Immunohistochemistry in Kidney
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Paraffin-embedded kidney tissues were sectioned at a thickness of 4 μm for IHC analysis. After dewaxing, rehydration, blocking, and antigen retrieval, the kidney tissue sections were exposed to anti-apelin antibodies (1:100) at 4 °C overnight. Then the sections were incubated with biotinylated goat anti-rabbit secondary antibodies (PV-9000, Zhongshan Jinqiao Biotechnology, Beijing, China) for 30 min at room temperature. A DAB kit (ZLI-9018, Zhongshan Jinqiao Biotechnology, Beijing, China) was used to detect the signal of the antigen-antibody complexes. Finally, the slides were counterstained in hematoxylin.
4
Apelin Immunohistochemistry in Kidney
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Paraffin-embedded kidney tissues were sectioned at a thickness of 4 μm for IHC analysis. After dewaxing, rehydration, blocking, and antigen retrieval, the kidney tissue sections were exposed to anti-apelin antibodies (1:100) at 4 °C overnight. Then the sections were incubated with biotinylated goat anti-rabbit secondary antibodies (PV-9000, Zhongshan Jinqiao Biotechnology, Beijing, China) for 30 min at room temperature. A DAB kit (ZLI-9018, Zhongshan Jinqiao Biotechnology, Beijing, China) was used to detect the signal of the antigen-antibody complexes. Finally, the slides were counterstained in hematoxylin.
5
IHC Staining Protocol for JMJD5 Expression
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JMJD5 expression in both tumor and normal tissues of the same lung cancer samples were determined by IHC staining. In brief, formalin fixed paraffin-embedded (FFPE) tumor tissues were cut into a series of 5µm thick sections. The sections were de-paraffinized and rehydrated. Antigen retrieval was achieved using Dako EnVisionTM FLEX Target Retrieval Solution (pH 9.0) in the Dako PT Link units at 95 °C for 20 minutes. Thereafter, each section was incubated with the anti-JMJD5 antibody in 1:100 (Rabbit polyclonal antibody, ab83011, Abcam) for 2 hours at room temperature. Followed by incubation with secondary antibody (PV-9000, Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) at room temperature for 30 minutes. Finally, the sections were colorized by 3,3'-diaminobenzamine and hematoxylin, respectively for immune complex and nuclei staining.
Both staining intensity and the percentage of positive tumor cells were evaluated as follows: (I) staining intensity: 0, absence of staining; 1, weak staining; 2, moderate staining; and 3, strong staining; (II) percentage of positive tumor cells: 0, 0–25% of staining; 1, 25–50% staining; 2, 50–75% staining; 3, ≥75% staining. The final score was obtained by multiplying the staining intensity score and percentage score. Final score ≥6 was defined as high expression.
Both staining intensity and the percentage of positive tumor cells were evaluated as follows: (I) staining intensity: 0, absence of staining; 1, weak staining; 2, moderate staining; and 3, strong staining; (II) percentage of positive tumor cells: 0, 0–25% of staining; 1, 25–50% staining; 2, 50–75% staining; 3, ≥75% staining. The final score was obtained by multiplying the staining intensity score and percentage score. Final score ≥6 was defined as high expression.
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