The expression of NRP2 of HCC tissues and matched adjacent normal liver tissues were examined by IHC. Paraffin sections of 4–5 μm were deparaffinized, and antigen was retrieved by boiling in 0.1 M citrate buffer (pH 6.0) for 20 minutes. Endogenous peroxidase was inactivated with 0.3% H2O2 for 10 minutes. Then, the primary antibody NRP2 (ab185710; dilution 1:100; Abcam, Cambridge, UK) was incubated for overnight at 4°C. PBS was used as a negative control. Subsequently, the sections were incubated with a biotinylated goat anti-rabbit secondary antibody for 30 minutes at a room temperature. Next, 3,3-diaminobenzidine was applied for 5 and 10 minutes. Each slide was counterstained with hematoxylin for redyeing. Then, the slides were dehydrated, making transparent and sealed.
Ab185710
Manufactured by Abcam
Sourced in United Kingdom, China
Anti-Ferritin heavy chain antibody (Ab185710) is a primary antibody that specifically binds to the heavy chain subunit of the ferritin protein. Ferritin is a ubiquitous intracellular protein that stores iron and releases it in a controlled fashion. The antibody can be used to detect and quantify ferritin levels in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab76943, by Abcam (1 mentions)
Ab81289, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Bca protein assay kit, by Qiagen (1 mentions)
Enhanced chemiluminescence reagent kit, by Beyotime (1 mentions)
Ab181602, by Abcam (1 mentions)
Bicinchoninic acid assay method, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer ripa buffer, by Beyotime (1 mentions)
3 protocols using ab185710
1
Immunohistochemical Analysis of NRP2 in HCC
2
Signaling Pathway Profiling by Western Blot
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Western blotting was performed with antibodies targeting the following proteins: VEGF receptor 2 (2479S, Cell Signaling), NRP2 (ab185710, Abcam), phospho-VEGF receptor 2 (Tyr951) (4991S, Cell Signaling), CD31 (ab28364, Abcam), CD34 (ab81289, Abcam), GAPDH (5174S, Cell Signaling), cofilin (5175S, Cell Signaling), phosphorylated cofilin (Ser3) (3313S, Cell Signaling), and SSH1 (ab76943, Abcam). Cells were lysed in RIPA buffer containing a protease inhibitor mixture (Roche) and incubated on a rocker at 4 °C for 15 min. The protein concentration of the lysates was measured using a BCA protein assay kit (Qiagen), and equal amounts of protein were separated by SDS-PAGE through 10% gels, transferred to PVDF membranes and probed with the indicated primary antibodies. Then, the blots were incubated with species-specific HRP-conjugated secondary antibodies, and the immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Pierce). Three independent experiments were performed.
3
Western Blot Analysis of NRP2 Expression
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Cells were washed twice with precold phosphate-buffered saline (PBS) and lysed in Radioimmunoprecipitation assay buffer (RIPA) buffer (Beyotime, Shanghai, China) on ice for 15 minutes. The protein concentration was quantified with the bicinchoninic acid assay method (Beyotime). Proteins were separated by 15% of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and subsequently transferred to the polyvinylidene difluoride membrane (Merck KGaA, Darmstadt, Germany). The membrane was blocked with 5% nonfat milk followed by incubating with primary antibody against NRP2 (ab185710; Abcam) or glyceraldehyde 3-phosphate dehydrogenase (ab181602; Abcam, Shanghai, China) at 4°C overnight. After washing twice with Tris-buffered Saline+Tween 20 (TBST), the membrane was incubated with horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature. The signals were visualized with the enhanced chemiluminescence reagent kit (Beyotime) according to the manufacturer’s instructions.
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