The HNEpCs were incubated until confluence on glass cover slips coated with poly-L-lysine (0.05%) in a growth medium for airway epithelial cells for 48 hours. Cells were pretreated with ghrelin (0.1 μM) with or without D-Lys-3-GHRP-6 (1 μM) for 1 hour. Pretreated cells were exposed to LPS (1 μg/mL) or leptin (0.1 μM) for 8 hours. The HNEpCs were fixed with formaldehyde (4%) in PBS for 15 minutes and blocked with BSA (5%) in PBS overnight. The HNEpCs were then incubated with MUC5AC antibodies (ab-198294; 1:100; Abcam). Subsequently, the samples were incubated with Alexa-488-labeled goat anti-rabbit IgG secondary antibodies. Nuclei were detected with 4´,6-diamidino-2-phenylindole (DAPI; AbCam). Stained samples were visualized by fluorescence microscopy (×40 magnification, Ti-S, 733551; Nikon). The intensity was measured using an infinite F200 PRO microplate fluorescence reader (Tecan).
Ab198294
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab198294 is a recombinant antibody produced in E. coli. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm800 confocal microscope, by Zeiss (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions)
Ab15481, by Abcam (1 mentions)
Ab92551, by Abcam (1 mentions)
Pas staining kit, by Merck Group (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
A21240, by Thermo Fisher Scientific (1 mentions)
T8787, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab68183, by Abcam (1 mentions)
Ab261733, by Abcam (1 mentions)
Ab220788, by Abcam (1 mentions)
Ab282575, by Abcam (1 mentions)
Ab2324, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab179467, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
7 protocols using ab198294
1
Ghrelin and LPS Regulation of MUC5AC in HNEpCs
2
Conjunctival Epithelial Characterization
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The ex vivo conjunctival cultures were fixed in 4% paraformaldehyde at 4 °C for 20 min, followed by a triple washing step with phosphate buffered saline. Stored mucin was detected using the PAS staining kit (Merck Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. Presence of MUC5AC (goblet cell marker) and MUC1 as well as CK13 (i.e., epithelial cell marker) were investigated by immunocytochemistry. Briefly, fixed cultures were permeabilized with 1% triton X-100 blocking buffer (30 min) and primary antibodies against MUC1 (Abcam (Cambridge, UK); ab15481, 1:200 dilution), MUC5AC (Abcam (Cambridge, UK); ab198294, 1:500 dilution), and CK13 (Abcam (Cambridge, UK); ab92551, 1:500 dilution) were incubated overnight at 4 °C. Cy3-conjungated donkey-anti-rabbit antibody (Jackson ImmunoResearch, Cambridge, UK) was added for 2 h at 4 °C, followed by a nuclear counterstain using 4′,6-diamidino-2-phenylindole (DAPI) for 1 min at room temperature. Samples were mounted in citifluor and imaged on an UltraVIEW VoX dual spinning disk confocal system (PerkinElmer, Billerica, MA, USA).
3
Immunostaining of Differentiated Nasal and Bronchial ALI Cultures
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Differentiated nasal and bronchial ALI cultures (n = 3) were fixed and stained as previously described [25 (link)]. Primary antibodies (β-tubulin IV (MU178-UC, Emergo Biogenex, Fremont, CA, USA), MUC5AC (ab198294, Abcam, Cambridge, UK), were incubated for 1 h at RT. Afterward, secondary antibodies (A-21240 and A11034, Invitrogen, Waltham, MA, USA), together with phalloidin (A34055, Mol. Probes, Eugene, OR, USA) and DAPI (D9542, Sigma, St. Louis, MO, USA), were added for 30 min. Both primary and secondary antibodies were diluted 1:500 in blocking buffer (1% BSA + 0.3 Triton X-100 (T8787, Sigma-Aldrich, St. Louis, MO, USA) in PBS). Images were acquired with a Zeiss LSM800 confocal microscope (40× objective). Image quantification was performed using Fiji (Max Planck Institute, Dresden, Germany) and CellProfiler (Broad Institute, Cambridge, MA, USA). Cultures did not show contamination with other cell types such as fibroblasts.
4
Protein Extraction and Western Blot Analysis
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Total protein was separated from the cell lysis buffer. For the detection of β-catenin in nuclei and cytoplasm, cells were collected and used to extract nuclear proteins and cytoplasmic protein, respectively (Biotechnology, Shanghai, China).The protein concentration was measured using the bicinchoninic acid (BCA) method (Beyotime). Proteins (20 µg) were separated via SDS polyacrylamide gels and then transferred onto PVDF membranes. After blocking for 2 h with 5% nonfat milk at room temperature, these membranes were incubated overnight with primary antibodies (MUC5AC, ab198294, 1/20,000; ICAM-1, ab282575, 1:1000; Bcl-2, ab32124, 1:1000; Bax, ab32503, 1:1000; cleaved caspase3, ab32042, 1:500; cleaved caspase9, ab2324, 1:1000; MMP10, ab261733, 1:1000; GAPDH, ab8245, 1:5000; β-catenin, ab68183, 1:1000; β-actin, ab179467, 1:5000; TBP, ab220788, 1:1000; Abcam, England). Membranes were washed three times by PBS and then incubated for 2 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc.).
5
Immunofluorescence Analysis of MUC5AC Expression in E-Cigarette Vapor-Treated Nasal Tissue
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To confirm the signaling pathway involved in e-cigarette vaporinduced MUC5AC expression in human nasal inferior turbinate tissue, we performed immunofluorescence (IF) staining in ex vivo culture. Before proceeding with the culture, the tissues were washed three times with PBS. After 8 hours of e-cigarette vapor (with or without nicotine 24 g/mL) treatment, tissue fragments were fixed in 4% paraformaldehyde, after which tissues were washed with PBS. Fixed tissues were embedded with optimal cutting temperature compound for 1 hour at −80°C. IF analyses were performed on 6-μm thick sections and blocked in 1% BSA in phosphate buffered saline with 0.1% Tween 20 for 1 hour at room temperature. The slides were incubated with a rabbit anti-MUC5AC antibody (ab-198294; Abcam, Cambridge, UK; 1:100 dilution). Subsequently, they were incubated with an Alexa 488-labelled goat anti-rabbit immunoglobulin G secondary antibody (green fluorescence). Nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; blue fluorescence, Abcam). The cells were visualized by fluorescence microscopy using Nikon software (Ti-S, 733551; Nikon, Tokyo, Japan).
6
Western Blot Analysis of Mucins and NF-κB Pathway
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After transfection, the treated or control cells were lysed with lysis buffer containing protease inhibitors. The protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to polyvinylidene fluoride membranes. After blocking with 5% defatted milk, the membranes were incubated with primary antibodies against MUC8 (Santa Cruz Biotechnology, Beijing, China), MUC5AC (ab198294, Abcam, USA), p65 (ab16502, Abcam), Lamin B (ab194109, Abcam), β-tubulin (ab6046, Abcam), IκBα (ab32518, Abcam), p-IκBα (ab133462, Abcam), and β-actin (ab8227, Abcam) at a dilution of 1 : 1000 at 4°C overnight. Following washing with Tris-Buffered Saline Tween-20 (TBST), the membranes were exposed to HRP-conjugated goat anti-rabbit (1 : 5000, Abcam) secondary antibodies. At last, protein bands were detected with an ECL detection system and quantified by ImageJ. Each experiment was repeated three times.
7
Fipronil Induces MUC5AC Expression in Human Nasal Tissue
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To confirm the signalling pathway involved in fipronil-induced MUC5AC expression in human nasal inferior turbinate tissue, we performed immunofluorescence (IF) staining in ex vivo culture. Tissues were cut into 2-to 3-mm 3 pieces under sterile conditions. Before proceeding with the culture, the tissues were washed 3 times with phosphate buffered saline (PBS). After 24 h of fipronil (10 µM) treatment, tissue fragments were fixed in 4% paraformaldehyde (PFA) in PBS for 24 h, after which tissues were washed with PBS. Fixed tissues were embedded with optimal cutting temperature (OCT) compound for 1 h at -80°C.
IF analyses were performed on 6-μm thick sections and blocked in 1 % BSA in PBST for 1 h at room temperature. The slides were incubated with a rabbit anti-MUC5AC antibody (ab-198294; Abcam, Cambridge, UK; 1:100 dilution). Subsequently, they were incubated with an Alexa 488-labelled goat anti-rabbit IgG secondary antibody (green fluorescence). Nuclei were stained with
IF analyses were performed on 6-μm thick sections and blocked in 1 % BSA in PBST for 1 h at room temperature. The slides were incubated with a rabbit anti-MUC5AC antibody (ab-198294; Abcam, Cambridge, UK; 1:100 dilution). Subsequently, they were incubated with an Alexa 488-labelled goat anti-rabbit IgG secondary antibody (green fluorescence). Nuclei were stained with
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