Tumor tissues collected from gastric cancer patients were fixed in 4.0% paraformaldehyde, embedded in paraffin and cut into 4-μm sections. Sections were incubated with 3.0% hydrogen peroxide to inactivate endogenous peroxidase and then blocked in 5.0% bovine serum albumin after antigen retrieval. To observe the relationship between GC-MSCs and TAM location within tumor, the sections were incubated at 4 °C overnight with primary antibodies against α-SMA (NBP2–33006, Novus Biologicals, Briarwood Avenue, CO, USA) and CD204 (bs136214, absin, Shanghai, China). Subsequently, incubation with Alexa Fluor 488-labelled goat anti-mouse IgG (H + L) and Alexa Fluor 555-labelled donkey anti-rabbit IgG (H + L) (Beyotime, Shanghai, China) was performed for 1 h at 37 °C in the dark. Nuclei were counterstained with DAPI (Beyotime). Fluorescent images were acquired by a confocal laser-scanning microscope (Ti2-E-A1, Nikon, Tokyo, Japan).
Nbp2 33006
Manufactured by Novus Biologicals
Sourced in United States
NBP2-33006 is a laboratory equipment product offered by Novus Biologicals. It is designed for use in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 labelled goat anti mouse igg h l, by Beyotime (1 mentions)
Ti2 e a1, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Azure 300 chemiluminescent imaging system, by Azure Biosystems (1 mentions)
Nb600 503, by Novus Biologicals (1 mentions)
Fitc labeled goat anti rabbit igg, by Beyotime (1 mentions)
6 diamidino 2 phenylindole dapi, by Merck Group (1 mentions)
Fv300, by Olympus (1 mentions)
Cy3 labeled goat anti rabbit igg, by Beyotime (1 mentions)
A0562, by Beyotime (1 mentions)
A0516, by Beyotime (1 mentions)
Nbp2 32831, by Novus Biologicals (1 mentions)
Nb600 408, by Novus Biologicals (1 mentions)
Triton x 100, by Beyotime (1 mentions)
C2687, by Merck Group (1 mentions)
Lsrfortessatm cell analyzer, by BD (1 mentions)
7 protocols using nbp2 33006
1
Immunofluorescent Visualization of GC-MSCs and TAMs in Gastric Cancer
2
Western Blotting of Pluripotent Stem Cells
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Protein samples for western blotting were isolated from culture-adapted PSCs by homogenization with lysis buffer (#9803, CST, USA). The samples were boiled in laemmli buffer (#161-0737, Bio-Rad, USA) for 5 min and were resolved on a 4%-12% gradient SDS-PAGE gel and blotted to PVDF membrane. Following overnight incubation with primary antibodies against α-SMA (1:1000; #NBP2-33006, Novus Biologicals, USA), GFAP (1:1000; #60190-1-Ig, Proteintech, USA) and beta-actin (1:1000, # NB600-503, Novus Biologicals, USA) and detection by HRP-conjugated IgG. Bands were visualized using an Azure 300 chemiluminescent imaging system (Azure Biosystems, USA).
3
Immunofluorescence Staining of LX2/HSCs
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LX2 cells or primary HSCs were seeded on glass coverslips. After the indicated treatment, cells were fixed with 4% paraformalde-hyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and then blocked with 5% bovine serum albumin in PBS for 1 h. After incubation with primary antibodies against NF-κB p65(1:100, NB100-82088, Novus Biologicals, Littleton, USA), α-SMA(1:100, NBP2-33006, Novus Biologicals), or desmin (1:100, NB120-15200, Novus Biologicals) overnight at 4 °C, cells were incubated with Cy3-labeled Goat Anti-Rabbit IgG(1:500, A0516, Beyotime, China) or FITC-labeled Goat Anti-Rabbit IgG (1:500, A0562, Beyotime) for another 1 h. Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and the images were captured using fluorescence microscopy (FV300, Olympus, Japan).
4
Quantifying Macrophage and Fibroblast Markers
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Cells were seeded at 40% confluence on round glass slides in 24-well plates. Cells were then fixed with 4% paraformaldehyde for 15 min at room temperature, followed by permeabilization with 0.1% Triton X-100 (Beyotime) in PBS. After blocking in goat serum at 37 °C for 30 min, the samples were incubated with antibodies against CD68 (Novus Biologicals, USA, NBP2-32831), CD206 (NovusBiologicals, USA, MAB2534), α-SMA (Novus Biologicals, USA, NBP2-33006), or Collagen I (Novus Biologicals, USA, NB600-408) in Diluent at 4 °C overnight. The samples were then incubated with secondary antibody (CST, USA, 8889/4412) at 37 °C for 1 h, followed by washing with PBS and staining with DAPI for nuclear visualization. Cell staining was examined under an inverted fluorescent microscope. The quantification of the mean fluorescence intensity (MFI) was performed using Image-Pro-Plus version 6.0.
5
Localization of Rbfox1 in Cells
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The localization of Rbfox1 in cells was determined through immunofluorescence staining. Briefly, freshly isolated MAs were fixed in 4% paraformaldehyde for 24 h at room temperature and then immersed in 15% sucrose and 30% sucrose for 10 min each step. Using a cryostation, the tissues were cut into 20-μm thickness. After permeabilizing with 0.2% Triton X-100 in PBS for 20 min, cells or tissues were blocked with 2% BSA in PBS for 40 min at room temperature. They were then incubated with rabbit anti-Rbfox1 (1 μg/mL, sc-135476, Santa Cruz Biotechnology) and mouse anti-α-smooth muscle actin (SMA) (0.4 μg/mL, NBP2-33006, Novus Biologicals) antibodies dissolved in the blocking solution at room temperature for 12 h. The information of antibodies was listed in Table S4 . After washing with PBS, cells or tissues were incubated for 1 h with secondary antibodies labeled with Alexa Fluor 488 or 594 (R37114 or R37119, Molecular Probes) at room temperature. A confocal laser scanning microscope (LSM710, Carl Zeiss, Germany) was used to capture the fluorescence signal.
6
Characterizing Smooth Muscle Cell Markers
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For flow cytometry analysis, SMCs were labeled with primary antibodies for anti-α-SMA (1:200, NBP2-33006, Novus biologicals, Centennial, CO, USA), anti-calponin (1:150, C2687, Sigma Aldrich, Buchs, Switzerland), anti-myostatin (1:50, H00002660-M07, Novus biologicals, Centennial, CO, USA) or anti-Isotype IgG mouse (1:150, sc-2877, Santa Cruz, Heidelberg, Germany). The cells were incubated with the secondary antibodies anti-mouse FITC (1:200, BD 55988, BD Biosciences, Allschwil, Switzerland) or anti-rabbit FITC (1:200, FP-SA5000 Alexa 488, Brunschwig, Switzerland). Analysis was conducted using an LSR FortessaTM cell analyzer (BD Bioscience, Allschwil, Switzerland) and results were analyzed with FlowJo software 7.2.4 (Tree Star Inc., Ashland, OR, USA).
7
Immunohistochemical Analysis of Laminin α1 and α-SMA
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Immunohistochemical staining was performed using the Ready-to-use HP IHC detection kit (cat. no. abs957; Absin Bioscience, Inc.). Briefly, the tissue sections were deparaffinized in xylene at room temperature and rehydrated with a descending alcohol series (100, 85 and 75%). For antigen retrieval, the tissue sections were immersed in sodium citrate at 100°C for 20 min followed by blocking in 3% hydrogen peroxide (from kit) and 100% FBS (Gibco; Thermo Fisher Scientific, Inc.) for 10 and 15 min, respectively, at room temperature. Subsequently, the sections were first incubated with primary antibodies against laminin α1 (1:200; cat. no. bs-4973R; BIOSS) and α smooth muscle actin (α-SMA; 1:200; cat. no. NBP2-33006; Novus Biologicals, Ltd.) at 4°C overnight and then with the corresponding secondary antibody provided by the HP IHC detection kit at room temperature for 2 h. The tissue sections were then stained with DAB reagent and hematoxylin for 2 min each at room temperature. Finally, the sections were observed under an optical photographic light microscope at ×200 magnification and the expression levels of laminin α1 and α-SMA were analyzed using Image Pro Plus 6.0 software.
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