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).
Yt2133
Manufactured by Immunoway
Sourced in United States
YT2133 is a laboratory instrument designed for the detection and analysis of various biomolecules. The core function of this equipment is to perform sensitive and accurate measurements using advanced spectroscopic techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ym3431, by Immunoway (2 mentions)
Yp0176, by Immunoway (2 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Ultrasensitive s p kit, by Maixin Group (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 yt2133
1
IHC Staining and H-Score Calculation for Protein Expression
2
Chromatin Immunoprecipitation and Dual-Luciferase Assays for HIF1A
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The SimpleChIP® Enzymatic Chromatin IP Kit (#9003, Cell Signaling Technology, USA) was utilized to perform ChIP assay according to the specification. In brief, CRC cells were fixed, collected and lysed. Then, pelleted nuclei by centrifugation followed by DNA digestion. This digested chromatin was incubated with HIF1A antibody (1:200 dilution, YT2133, Immunoway, USA) and magnetic beads to perform immunoprecipitation. The chromatin was eluted from the magnetic beads and detected using PCR assay. The Dual Luciferase Reporter Assay System (E1910, Promega, WI, USA) was used to perform dual-luciferase reporter assay according to the instruction and previous research [14 (link)]. HIF1A siRNA and HIF1A activator ML228 (5 μM, HY-12754, MCE, China) were used in the dual-luciferase reporter assay.
3
Immunohistochemical Analysis of HIF-1α, VEGF-A, and Ki67
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The expression of HIF-1α, VEGF-A, and Ki67 was detected by conventional immunohistochemical (IHC) staining protocol. In brief, formalin-fixed paraffin-embedded tumor tissue blocks were cut into 4 μm thick sections. After drying, de-paraffinized, and dehydrated in graded ethanol, tissue sections were immersed in 3% hydrogen peroxide for 10 minutes at room temperature to block endogenous tissue peroxidase activity. The antigen was retrieved by citrate buffer (pH 6.0) and high-heat microwave processing for 5 minutes, followed by washing in PBS. All slides were incubated with primary HIF-1α antibody (YT2133, Immunoway, 1:200 dilution), VEGF-A antibody (YT5108, Immunoway, 1:200 dilution), and Ki67 antibody (SP6, DAKO, ready-to-use) overnight at 4°C, followed by incubation for 30 minutes in Ultra-Sensitive SP kit (Maixin-Bio, Fuzhou, China). The slides were rinsed with phosphate-buffered saline before color development using a 3, 3’-diaminobenzidine substrate kit and were then counterstained with hematoxylin.
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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