At 48 h post-infection, HBE cells were fixed in 4% PFA for 72 h, washed with ice cold PBS, and blocked with 1% bovine serum albumin (BSA) + 10% normal goat serum in PBS + 0.05% Tween-20 (PBS-T). Antibodies used included acetylated-α-tubulin (EMD Millipore #MABT868, 1:100), MUC5AC (clone 45M1 Sigma Aldrich #M5293, 1:100) and zonula occludens-1 (ZO-1 Invitrogen #339194 conjugated to AlexaFluor-594, 1:100) diluted in blocking buffer were added for 3 h at 37°C (whole filter) or 24h at 4°C (cross-sections). Following three washes with PBS and incubation with blocking buffer for 10 min, cells were incubated with secondary antibody α-mouse IgG AF594 (Life Technologies #A11020, 1:500) for 1.5 h at 37°C. Cells were then washed with PBS, and Transwell filters were mounted on glass slides with Prolong Gold antifade with DAPI (Invitrogen). Cells were imaged with an Echo Revolve R4 immunofluorescent microscope (Echo). Quantification of fluorescence was performed using ImageJ software (v1.52a). For each bacterial strain, two biological experiments in technical duplicate were performed.
Mabt868
Manufactured by Merck Group
Sourced in United States
MABT868 is a laboratory instrument designed for specialized analytical tasks. It features precise measurement capabilities and advanced data processing functionalities. The core function of MABT868 is to perform high-accuracy analyses on a variety of samples.
Automatically generated - may contain errors
Lab products found in correlation
A 21311, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
A 21235, by Thermo Fisher Scientific (1 mentions)
Infinity5 camera, by Teledyne (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Csu w1 spinning disk confocal microscope, by Nikon (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
A32727, by Thermo Fisher Scientific (1 mentions)
6 protocols using mabt868
1
Immunofluorescence Analysis of HBE Cells
2
Whole-mount Immunostaining of Embryos
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Euthanized embryos were fixed in a solution containing 4% paraformaldehyde in PBS for at least 2 h at room temperature. Following fixation, samples were washed with 0.3% PBSTx ( min), permeabilized with acetone (100% acetone, 10 min incubation at C), washed with 0.3% PBSTx ( min) and blocked in 0.1% BSA/0.3% PBSTx for 2 h. Embryos were incubated with acetylated tubulin antibody (clone 6-11B-1, MABT868, Sigma-Aldrich, 1:1000) overnight at C. On the next day samples were washed (0.3% PBSTx, h) and incubated with the secondary antibody (Alexa-labeled goat-anti-mouse 555 plus, A32727, Thermo Fisher Scientific, 1:1000), an anti-GFP antibody conjugated with Alexa 488 (A-21311, Thermo Fisher Scientific, 1:1000) and DAPI (D1306, Thermo Fisher Scientific, 1:1000) overnight at C. The next day samples were washed (0.3% PBSTx, h) and transferred to a series of increasing glycerol concentrations (25%, 50%, and 75%).
3
Spatiotemporal Expression Analysis of Developmental Genes
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Fragments of chordin and Hox genes were isolated as previously described24 (link) using gene-specific oligonucleotides and a T7 adaptor. Riboprobes were synthesized using a T7 MEGAscript kit (ThermoFisher, AM1334) and stored at a concentration of 50 ng µl–1 in hybridization buffer at –20 °C. Whole-mount in situ hybridization in embryonic, larval and juvenile stages were conducted as described elsewhere24 (link),26 (link). Antibody staining in larval stages of O. fusiformis, Magelona spp. and C. teleta was carried out as previously described23 (link),115 (link) using the following antibodies: mouse anti-acetyl-α-tubulin antibody, clone 6-11B-1, 1:800 dilution (Sigma-Aldrich, MABT868, RRID: AB_2819178) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 647, 1:800 dilution (Thermo Fisher Scientific, A-21235, RRID: AB_2535804). Differential interface contrast images of the colorimetric in situ were obtained using a Leica 560 DMRA2 upright microscope equipped with an Infinity5 camera (Lumenera). Fluorescently stained samples were scanned using a Nikon CSU-W1 spinning disk confocal microscope.
4
Immunostaining of Kupffer's Vesicle Cilia
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Embryos were fixed at the 8-somite stage using 4% paraformaldehyde at 4°C overnight. Anti-acetyl-alpha-tubulin antibody (1:500, Sigma, MABT868, USA) was used to label the cilia in Kupffer's vesicles (KVs). Immunofluorescence staining was performed as previously described (33 (link)). Before permeation with acetone, embryos were equilibrated in Tris–HCl (0.1 M, pH 9.0) for 5 min at room temperature and then heated at 70°C for 15 min for antigen retrieval. After staining, embryos were flattened and imaged using the z-stack function of the confocal microscope with a ×60 oil objective.
5
KRAS and PDE6D Transfection and Imaging
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The NIH-3T3 cells were transfected
with KRAS and PDE6D constructs using Lipofectamine 2000 following
the provider’s guidelines. After transfection, the cells were
incubated overnight for 24 h and transferred onto 1.5-thickness glass
coverslips (VWR, Avantor, Radnor, PA, US) and allowed to attach overnight.
Next, the cells were starved with 0.5% calf serum-containing media
for 24 h and fixed with 4% paraformaldehyde (Thermo Fisher Scientific)
for 20 min. After fixation, the coverslips were extensively washed
with phosphate-buffered saline (PBS) and the cells were permeabilized
using 0.3% Triton X-100 (Sigma/Merck) for 5 min at room temperature.
After washing in PBS, the samples were blocked using 2% bovine serum
albumin (BSA) (Sigma/Merck) for at least 30 min, followed by antiacetylated
tubulin MABT868 (Sigma) overnight at 4 °C. After extensive washing
in PBS, the cells were incubated with goat antimouse IgG H + L antibody
Abberior Star Red from Abberior (GmbH, Göttingen, Germany)
for 45 min in darkness at room temperature. The coverslips were mounted
onto glass slides using fluoromount-G (Southern Biotech, Birmingham,
AL, USA) and cured for at least 6 h before imaging.
with KRAS and PDE6D constructs using Lipofectamine 2000 following
the provider’s guidelines. After transfection, the cells were
incubated overnight for 24 h and transferred onto 1.5-thickness glass
coverslips (VWR, Avantor, Radnor, PA, US) and allowed to attach overnight.
Next, the cells were starved with 0.5% calf serum-containing media
for 24 h and fixed with 4% paraformaldehyde (Thermo Fisher Scientific)
for 20 min. After fixation, the coverslips were extensively washed
with phosphate-buffered saline (PBS) and the cells were permeabilized
using 0.3% Triton X-100 (Sigma/Merck) for 5 min at room temperature.
After washing in PBS, the samples were blocked using 2% bovine serum
albumin (BSA) (Sigma/Merck) for at least 30 min, followed by antiacetylated
tubulin MABT868 (Sigma) overnight at 4 °C. After extensive washing
in PBS, the cells were incubated with goat antimouse IgG H + L antibody
Abberior Star Red from Abberior (GmbH, Göttingen, Germany)
for 45 min in darkness at room temperature. The coverslips were mounted
onto glass slides using fluoromount-G (Southern Biotech, Birmingham,
AL, USA) and cured for at least 6 h before imaging.
6
Immunofluorescent Labeling of Embryos
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Euthanized embryos were fixed in a solution containing 4% PFA in PBS for at least 2h at room temperature. Following fixation, samples were washed with 0.3% PBSTx (3 x 5 min), permeabilized with acetone (100% acetone, 10 min incubation at -20°C), washed with 0.3% PBSTx (3 x 10 min) and blocked in 0.1% BSA/0.3% PBSTx for 2 h. Embryos were incubated with acetylated tubulin antibody (clone 6-11B-1, MABT868, Sigma-Aldrich, 1:1000) overnight at 4 °C. On the next day samples were washed (0.3%PBSTx, 3 x 1 h) and incubated with the secondary antibody (Alexa-labelled goat anti-mouse 555 plus, A32727, Thermo Fisher Scientific, 1:1000), an anti-GFP antibody conjugated with Alexa 488 (A-21311, Thermo Fisher Scientific, 1:1000) and DAPI (D1306, Thermo Fisher Scientific, 1:1000) overnight at 4 °C. The next day samples were washed (0.3% PBSTx, 3 x 1 h) and transferred to a series of increasing glycerol concentrations (25%, 50% and 75%).
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