IHC staining and H-Score calculating were performed as our previous research [17 (link)]. The primary antibodies used in the assay were anti-RRAGB (1:300, 13,023–1-AP, Proteintech, USA), anti-p-mTOR (Ser2448) (1:200, YP0176, Immunoway, USA), anti-HIF1A (1:300, YT2133, Immunoway, USA), and anti-cleaved-caspase3 (1:100, YM3431, Immunoway, USA).
Yp0176
Manufactured by Immunoway
Sourced in United States
YP0176 is a laboratory equipment designed for centrifugation. It is a compact and efficient device used for the separation of different components within a liquid sample.
Automatically generated - may contain errors
Lab products found in correlation
Yt2133, by Immunoway (2 mentions)
Ym3431, by Immunoway (2 mentions)
Rm2135, by Leica (1 mentions)
Citrate buffer ph 6, by Solarbio (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Hematoxylin, by Solarbio (1 mentions)
Ab32150, by Abcam (1 mentions)
Ab191217, by Abcam (1 mentions)
Ab32064, by Abcam (1 mentions)
Bs13278, by Bioworld Technology (1 mentions)
Bs12478, by Bioworld Technology (1 mentions)
Dura ecl kit, by Fdbio (1 mentions)
Yt3555, by Immunoway (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
4 protocols using yp0176
1
IHC Staining and H-Score Calculation for Protein Expression
2
Immunohistochemical Analysis of Xenograft Tumors
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Tissue samples isolated from xenograft tumors were subjected to histological analysis. Tumor samples were first fixed with formalin and then embedded in paraffin, cut into 5 µm sections, and stained with H&E for immunohistochemistry (IHC) staining. The primary antibodies used were anti-LC3 (1:1000, 14600-1-AP; Proteintech), anti-p-mTOR (1:1000, YP0176; Immunoway), and anti-phospho-Akt (1:1000, #4060; CST). Images were visualized using an Olympus inverted microscope (Olympus Corp.), and image analysis was performed by ImageJ.
3
Immunohistochemical Analysis of p-mTOR in Hippocampus
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Staining of p-mTOR (S2448) was performed, as follows: Following the embedding of the hippocampal tissues in paraffin (Shanghai Hualing Recovery Appliance Factory, Shanghai, China), sections (5 µm) from the CA3 area were transferred to slides (Leica RM 2135, Leica Microsystems GmbH, Wetzlar, Germany) and deparaffinized. The slides were then incubated with citrate buffer (pH 6; Beijing Solarbio Science & Technology Co., Ltd.) for 5 min in a microwave oven twice (with a 10 min interval between) and cooled to room temperature. The slides were then incubated with rabbit p-mTOR polyclonal antibody (Ser2448; 1:65; YP0176; ImmunoWay Biotechnology Company, Newark, DE, USA) at 4°C overnight, and then in the anti-rabbit secondary immunoglobulin (Ig)G antibody conjugated to horseradish peroxidase (PV6001; OriGene Technologies, Inc., Beijing, China) for 30 min. A 3,3′-diaminobenzidine kit (ZLI-9017; OriGene Technologies, Inc.) was used for visualization, and slides were counterstained with hematoxylin (Beijing Solarbio Science & Technology Co., Ltd.). The average integrated optical density value was obtained by analyzing five fields per slide using Image-Pro Plus software (v. 6.0; Media Cybernetics, Carlsbad, CA, USA).
4
Quantitative Protein Analysis by Western Blot
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The total protein was separated by SDS-PAGE electrophoresis and transferred onto PVDF membrane (#IPVH00010, Millipore, USA). Then, the PVDF membrane was incubated with the following primary antibodies at 4 °C overnight: anti-RRAGB (1:1000, 13,023–1-AP, Proteintech, USA), anti-mTOR (1:500, 66,888–1-IG, Proteintech, USA), anti-p-mTOR (Ser2448) (1:1000, YP0176, Immunoway, USA), anti-S6K (1:1000, YT3555, Immunoway, USA), anti-p-S6K (Thr389) (1:1000, YP1427, Immunoway, USA), anti-HIF1A (1:1000, YT2133, Immunoway, USA), anti-pro-caspase3 (1:1000, AB32150, Abcam, USA), anti-cleaved-caspase3 (1:500, YM3431, Immunoway, USA), anti-PARP (1:1000, AB191217, Abcam, USA), anti-cleaved-PARP (1:1000, AB32064, Abcam, USA), and anti-β-tubulin (1:2000, 10,094–1-AP, Proteintech, USA). Subsequently, the PVDF membrane was incubated with the corresponding HRP labeled goat anti-mouse (1:500, BS12478, Bioworld, USA) or goat anti-rabbit IgG (1:500, BS13278, Bioworld, USA) at room temperature for 1 h. FDBio-Dura ECL Kit (FD8020, FDbio, China) was used to visualize the protein bands.
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