For immunofluorescence staining, microfluidic devices were washed with 1x PBS and perfused with 0.2% TritonX-100 for 2 min on ice. Subsequently, the chambers were washed with 1x PBS and incubated with 4% PFA for 20 min at RT. Afterwards, the devices were washed with 1x PBS and further permeabilized with 0.05% TritonX-100 for 5 min on ice. Cells were incubated for 1 h at RT with the following primary antibodies: goat anti-human zonula occludens (ZO)-1 (1:100; Invitrogen) and mouse anti-human e-cadherin (1:100; BD Biosciences). Afterwards, the chambers were washed with PBS and blocked with 1% BSA in 1x PBS for 1 hr at RT. The devices were washed with 1x PBS and incubated for 1 h with following secondary antibodies: Alexa-488 donkey anti-mouse IgG (1:1000; Thermo Fisher Scientific), Alexa-647 donkey anti-goat IgG, (1:1000; Thermo Fisher Scientific). Cell nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI)(1:1000; BD Biosciences). As the last step, the devices were washed with 1x PBS and filled with fresh 1x PBS. Micrographs were obtained with an inverted fluorescence microscope (IX-83, Olympus) using 10× and 20× long distance objectives. Images were analyzed using ImageJ.
Mouse anti human e cadherin
Manufactured by BD
Sourced in United States
Mouse anti-human E-cadherin is a laboratory reagent used for the detection and quantification of E-cadherin, a cell-cell adhesion protein, in human samples. It is designed for use in various analytical techniques, such as immunohistochemistry and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Rabbit anti human cleaved caspase 3, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole dapi, by BD (1 mentions)
Axiovert microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole dapi staining, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Emr8 5, by Abcam (1 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Streptavidin hrp, by R&D Systems (1 mentions)
Pierce protease inhibitor mini tablet, by Thermo Fisher Scientific (1 mentions)
Mouse anti human β actin, by Abcam (1 mentions)
Mem per plus protein extraction kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using mouse anti human e cadherin
1
Immunofluorescence Staining of Cell-Laden Microfluidic Devices
2
Immunocytochemical Localization of CTSB in MCF-7 Cells
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For immunocytochemical staining of CTSB, MCF-7 cells were seeded at a density of 20 × 103 on glass cover-slips in RPMI-1640 supplemented with 3% FBS and different concentrations of recombinant IL-6 (10 ng/mL, 25 ng/mL and 50 ng/mL) for 48 h. To immunolocalize intracellular CTSB, MCF-7 cells seeded in different concentrations of IL-6 were washed four times with PBS and fixed with methanol (−20 °C) for 5–10 min. Cells were then permeabilized with 2% saponin in PBS and blocked by incubating with 0.5% bovine serum albumin in PBS. CTSB intracellular staining was carried out as previously described [29] (link). The primary antibodies used were rabbit anti-human CTSB (5 μg/mL), mouse anti-human E-cadherin (BD Bioscience CA, USA) and rabbit anti-human vimentin (Santa Cruz Biotech, CA, USA). The secondary antibodies used were fluorescein-conjugated affinity-purified donkey anti-rabbit IgG and Texas red-conjugated affinity-purified donkey anti-mouse IgG (20 μg/mL) in 5% normal donkey serum. Cell nuclei were visualized by 4′,6′-diamidino-2-phenylindole (DAPI) staining (Sigma, Deisenhofen, Germany). Control samples were run in parallel but the primary antibody was omitted. Coverslips were mounted with anti-fade reagent and were analyzed with a Zeiss Axiovert microscope (Zeiss, Oberkochen, Germany) at 40X magnification.
3
Multiparametric Flow Cytometry of Apoptosis and Cell Surface Markers
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Cells were rinsed with warm PBS and harvested with a non-enzyme cell dissociation buffer (Life Technology). After centrifugation, cells were fixed with 2% paraformaldehyde in PBS for 30 min then rinsed with 1% FBS/PBS. For total expression, cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min and subsequently washed in PBS. Both membranous and total expression samples were then incubated with an FcR Blocker along with the primary antibodies in 1% FBS/PBS for 30 min at 4 °C. They were then washed with 1% FBS/PBS and incubated with secondary antibodies for 30 min at 4 °C. The primary antibodies used were rabbit anti-human cleaved caspase-3 (9661, Cell Signaling Technology), mouse anti-human E-cadherin (Clone 36, BD), mouse anti-human HLA Class 1 ABC antibody (EMR8-5, Abcam) and rabbit anti-human PD-L1 (28-8, Abcam). Secondary antibodies used were goat anti-mouse Alexa Fluor® 647 or goat anti-rabbit Alexa Fluor® 488 (Life Technologies). Cells were sorted on BD FACSCalibur™ and analyzed with FlowJo (v10.6.2).
4
Immunofluorescence Staining of Adherent Cells
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Cells on coverslips were fixed in cold 2% paraformaldehyde in PBS, and then permeabilized in 0.2% Triton X-100 in PBS for 20 min. Cells were subsequently washed in PBS, blocked for 1 h at room temperature (RT) in PBS containing 2% bovine serum albumin (Sigma-Aldrich). Fixed cells were then incubated with the primary antibody in 2% BSA/PBS solution overnight at 4 °C. Cells were washed in PBS three times, and the secondary antibody was added in 2% BSA/PBS solution for 1 h at RT in the dark. Cells were washed three times in PBS and then incubated for 2 min in Hoechst 33342 solution, after which they were washed in PBS and mounted. Confocal images were obtained on an Olympus upright Fluoview 2000 confocal microscope (Center for Biologic Imaging, University of Pittsburgh, supported by NIH #1S10OD019973-01) using a 60x (UPlanApo NA = 1.42) or 20x (UPlanSApo NA = 0.85) objective. The primary antibodies used were rabbit anti-human cleaved caspase-3 (9661, Cell Signaling Technology), mouse anti-human E-cadherin (Clone 36, BD), mouse anti-human HLA Class 1 ABC antibody (EMR8-5, Abcam) and mouse anti-human PD-L1 (28-8, Abcam). Secondary antibodies used were goat anti-mouse Alexa Fluor® 488 or goat anti-rabbit Alexa Fluor® 647 (Life Technologies). Hoechst 33342 was applied for nuclei counterstaining.
5
Biotinylated Factor H Binding Study
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Monocytes were incubated for 30 min in PBS with or without biotinylated FH. Cells were lysed with NP-40 lysis buffer supplemented with Pierce protease inhibitor mini tablets (#88666; Thermo Scientific) and Halt phosphatase inhibitor cocktail (#78420; Thermo Scientific). Cell fractionation was performed using Mem-PER Plus protein extraction kit (#89842; Thermo Scientific). FH was detected with Streptavidin-HRP (#DY998; R&D Systems) and endosomes with rabbit anti-human EEA1 antibody (#PA1-063A; Invitrogen). For fractionation controls, mouse anti-human β-actin (#ab8226; Abcam) and mouse anti-human E-cadherin (#610182; BD) were used.
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