Cells were cultured on different stiffness of PA hydrogel in 24-well plates, after indicated treatments, cells were fixed in 4% paraformaldehyde for 15 min and washed three times with PBS. Next, cells were permeabilized treatment and blocked using 0.25% Triton X-100 and 0.5% BSA in PBS for 30 min, then washed three times with PBS. Then, cells were probed with primary anti-YAP antibodies (dilution, 1:300; Abcam, USA) diluted in 1% BSA/PBS overnight at 4 °C After five washes in PBS, cells were incubated with Alexa Fluor 488-labeled secondary antibodies (dilution, 1:500; Beyotime Biotechnology, Shanghai, China) diluted in 1% BSA/PBS for 1 h at room temperature. After five washes in PBS, nuclear was stained with 4′, 6′-diamidino-2-phenyl-indole dihydrochloride (DAPI; Solarbio, Beijing, China). After five washes in PBS, PA hydrogel were taken out from 24-well plate and placed on glass slide. Images were acquired using confocal microscope.
Alexa fluor 488 labeled secondary antibody
Manufactured by Beyotime
Sourced in China
The Alexa Fluor 488-labeled secondary antibody is a fluorescently-conjugated antibody that binds to the primary antibody, allowing for the detection and visualization of target molecules in various assays and applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (2 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Beyotime (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Beyotime (2 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Solarbio (1 mentions)
D7679msds, by Merck Group (1 mentions)
Fv10i laser confocal microscope, by Olympus (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica camera (1 mentions)
Malondialdehyde mda assay kit, by Nanjing Jiancheng (1 mentions)
3 3 4 5 tms, by Tokyo Chemical Industry (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt griess reagent, by Merck Group (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Saccharomyces cerevisiae strain ah109, by Takara Bio (1 mentions)
Ypd medium, by Takara Bio (1 mentions)
M iggκ bp hrp, by Santa Cruz Biotechnology (1 mentions)
C caspase 3, by Beyotime (1 mentions)
Hrp conjugated secondary antibody, by Beyotime (1 mentions)
9 protocols using alexa fluor 488 labeled secondary antibody
1
Immunostaining of YAP in Cells on PA Hydrogels
2
Immunohistochemical Analysis of Esophageal Tumors
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Esophageal tumors and normal esophageal tissues (tumor free individuals) from patients were fixed with 10% formaldehyde for 2 h at 37°C, then embedded in paraffin and cut into tumor 4 µm thick sections. Antigen retrieval was performed on the tumor sections using eBioscience™ IHC Antigen Retrieval Solution (cat no. 00-4955-58, Invitrogen; Thermo Fisher Scientific, Inc.), sections were washed with PBST (Sigma-Aldrich; Merck KGaA) and subsequently incubated with mouse anti-human FGFR (1:1,000, cat no. ab10646, Abcam) or mouse anti-human VEGFR antibodies (1:1,000, cat no. ab2349, Abcam) for 12 h at 4°C. Following antibody incubation, proteins were washed with PBST three times and incubated with Alexa Fluor 488-labeled secondary antibodies (1:500; Beyotime Institute of Biotechnology, Haimen, China) for 2 h at 37°C and the specimens were visualized. A Diaminobenzidine staining system (D7679MSDS, Sigma-Aldrich; Merck KGaA) was used to detect target protein expression according to manufacturer's protocol. For histological staining, tumor sections were stained with hematoxylin and eosin and observed using a light microscope (Olympus BX51, Olympus Corporation, Tokyo, Japan) as described previously (34 (link)).
3
Cellular Uptake of Labeled sEVs
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When LNCaP cells confluenced to 50–60%, LNCaP-AI + F sEVs labeled with the lipophilic phospholipid membrane dye Dil (Beyotime, China) were added and co-cultured for 48–72 h. After removal of the cell culture medium, the cells were gently washed with PBS three times, fixed with 4% paraformaldehyde for 20 min followed by an overnight incubation with E-cadherin primary antibody (Beyotime, China). The next day, cells were washed twice with PBS and incubated with Alexa Fluor 488-labeled secondary antibody (Beyotime, China) at room temperature for 2 h. Cells were washed again and then stained with DAPI for 5 min. The samples were observed on the FV10i laser confocal microscope (Olympus, Japan) with a 60x UIS2 SAPO air objective.
4
Macrophage Activation and NF-κB Translocation
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RAW264.7 macrophages (3 × 105 cells/well) were seeded into confocal dish and incubated overnight (5% CO2, 37 °C). Cells were pretreated with daidzin (50 µM), daidzein (50 µM) and DXMS (5 µM, positive control) for 4 h and then exposed to LPS (1 µg/mL) for 4 h to stimulate cells. After washing 3 times with 1× phosphate-buffered saline (PBS), the cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min. The residual PFA was washed away with PBS. 0.1% Triton X-100 was used to permeabilize the fixed cells for 10 min. The cells were blocked with 3% BSA for 30 min at room temperature and then incubated with the primary antibody for p65 (1:200) overnight at 4 °C, followed by a 1-h incubation with Alexa Fluor 488-labeled secondary antibody (1:100, Beyotime Biotechnology) at 37 °C incubator. After washing with PBS, the cell nuclei were stained by DAPI (Beyotime Biotechnology). The fluorescence images were captured by Leica TCS SP8 confocal laser scanning microscope.
5
Assessing Cellular Antioxidant and Inflammatory Responses
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3,3′,4,5′-TMS and 3,4′,5-TMS (purity > 98%) were purchased from Tokyo Chemical Industry Co., Ltd. High-glucose Dulbecco’s modified Eagle’s medium (DMEM) were obtained from GE Healthcare Life Sciences HyClone Laboratories (Utah, USA) while fetal bovine serum (FBS) and Penicillin-streptomycin (P/S) were from Gibco (Carlsbad, CA, USA). LPS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Griess reagent, bovine serum albumin (BSA) and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 and TNF-α were purchased from Mlbio (Shanghai, China). All the primary antibodies were purchased from Cell Signaling Tech (Danvers, MA, USA) while the secondary antibodies were obtained from Beyotime Biotechnology (Shanghai, China). 4% paraformaldehyde (PFA), Alexa Fluor 488-labeled secondary antibody, and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were purchased from Beyotime Biotechnology (Shanghai, China). Dihydroethidium (DHE) and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) were acquired from Molecular Probes (Eugene, OR, USA). Malondialdehyde (MDA) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
6
Protein expression and purification protocol
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Anti-His antibody-HRP was purchased from Thermo Fisher (Waltham, MA, USA). Saccharomyces cerevisiae strain AH109, YPD medium and yeast selective culture medium were purchased from Clontech (Palo Alto, CA, USA). T4 DNA ligase and restriction enzymes were purchased from Takara Biomedicals (Kyoto, Japan). pGBKT7-CXCR4 was constructed and optimized by Genewiz (Suzhou, China). E. coli strains (DH5a, BL21) and Ni-MAC Cartridge were from Novagen. Primary antibodies against Bax, Bcl-2, cleaved caspase-8 (c-caspase-8), cleaved caspase-3 (c-caspase-3), cleaved-PARP-1 (c-PARP-1), HRP-conjugated secondary antibody, and Alexa Fluor 488-labeled secondary antibody were from Beyotime Biotechnology (Shanghai, China). Primary antibody against pro-caspase-9 was from ZFdows BIO (Nanjing, China). Primary antibody against p-p53 and secondary antibody m-IgGκ BP-HRP were from Santa Cruz Biotechnology (CA, USA). Primary antibody against β-actin was from ZSGB-BIO (Beijing, China).
7
Immunofluorescent Staining of Cells
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Immunofluorescent staining was conducted as described previously.52 (link) Briefly, at the indicated time points, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 (Beyotime, Shanghai, China) for 15 min followed by blocking with 10% goat serum (Beyotime, Shanghai, China) at room temperature for 1 h. Then, cells were incubated with the primary antibodies overnight at 4°C and then incubated with Cy3-labeled secondary antibody (BOSTER, Wuhan, China) or Alexa Fluor 488-labeled secondary antibody (Beyotime, Shanghai, China) for 1 h at 37°C in the dark. Subsequently, cells were stained with 4,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China) for 10 min. Five random fields at 400 × magnification were photographed under a fluorescent microscope (Olympus BX53, Tokyo, Japan).
8
Evaluating NF-κB p65 Translocation in bEnd.3 Cells
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bEnd.3 cells (5 × 105/confocal dish) cultured in a laser confocal Petri dish were pretreated with piceatannol for 30 min, followed by the co-incubation of LPS (10 μg/mL) for 1 h. The bEnd.3 cells were then washed by PBS, fixed with 4% PFA for 20 min, permeabilized with 1% Triton X-100 for 10 min and blocked with 3% BSA for 1 h at room temperature. Subsequently, the cells were incubated with primary antibody against NF-κB p65 (1:500) (#8242, Cell Signaling Technology) overnight at 4 °C, followed by incubation with Alexa Fluor 488-labeled secondary antibody (1:100) (A0423, Beyotime) for 2 h at room temperature. Finally, the cells were washed with PBS and subjected to DAPI incubation. The fluorescence images were captured by a confocal laser scanning microscope (Leica Microsystems, TCS SP8, Mannheim, Germany). The quantification (measurement of intensity) was performed using the Image-Pro Plus 6.0 software.
9
Piceatannol Modulates Inflammatory Response
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Piceatannol was purchased from Tokyo Chemical Industry (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) was bought from GE Healthcare Life Sciences HyClone Laboratories (Logan, UT, USA), while fetal bovine serum (FBS) and penicillin plus streptomycin (PS) were obtained from Gibco (Carlsbad, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). The secondary antibodies, RIPA buffer, phenyl methane sulfonyl fluoride (PMSF), Protease Inhibitor Cocktail, BCA protein assay kit for Western blotting, 4% paraformaldehyde (PFA), Alexa Fluor 488-labeled secondary antibody, and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were acquired from Beyotime biotechnology (Shanghai, China), while all the primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). PVDF membranes were acquired from Millipore (Billerica, MA, USA). 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) was purchased from Molecular Probes (Eugene, OR, USA).
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