Forty-eight hours after transfection, HeLa cells were processed for immunofluorescence. The cells were washed twice with phosphate-buffered saline (PBS: 140 mM NaCl, 2 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4 pH 7.4) and fixed for 20 minutes with 3,7% formaldehyde in PBS. Cells permeabilization was performed by 20 minutes incubation with 0,1% Triton X-100 in PBS, followed by 30 minutes wash with 1% gelatin (type IV, from calf skin, Sigma) in PBS. The coverslips were then incubated for 60 minutes at 37 °C in a wet chamber with a rabbit polyclonal anti-PMCA3 antibody (PA1916, Thermo Fisher) at a 1:100 in PBS. Staining was revealed by the incubation with specific AlexaFluor 488-labeled anti-mouse secondary antibody, 1:50 (A-11001, Life technologies) for 45 minutes at room temperature. Fluorescence was detected with a Leica SP5 confocal microscope and analysed by ImageJ software.
Alexa fluor 488 labeled anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in China
The Alexa Fluor 488-labeled anti-mouse secondary antibody is a fluorescent-labeled detection reagent. It is designed to bind to primary antibodies raised in mice, allowing for the visualization and detection of target proteins or molecules in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Sp5 confocal microscope, by Leica (2 mentions)
Gelatin type 4, by Merck Group (2 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Pa1916, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti pmca antibody, by Thermo Fisher Scientific (1 mentions)
Prolong gold anti fade reagent containing dapi, by Thermo Fisher Scientific (1 mentions)
Anti ha 11, by BioLegend (1 mentions)
Prolong antifade mountant, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
Fluoromount g mounting medium, by Southern Biotech (1 mentions)
5 protocols using alexa fluor 488 labeled anti mouse secondary antibody
1
Immunofluorescence Staining of PMCA3 in HeLa Cells
2
Visualizing PMCA in HeLa Cells
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Forty-eight hours after transfection, HeLa cells were processed for immunofluorescence. The cells were washed twice with phosphatebuffered saline (PBS: 140 mM NaCl, 2 mM KCl, 1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 pH 7.4) and fixed for 20 min with 3,7% formaldehyde in PBS. Cells permeabilization was performed by 20 min incubation with 0,1% Triton X-100 in PBS, followed by 30 min wash with 1% gelatin (type IV, from calf skin, Sigma) in PBS. The coverslips were then incubated for 60 min at 37 °C in a wet chamber with a mouse monoclonal anti-PMCA antibody, 1:20 (Thermo Scientific) in PBS. Staining was revealed by the incubation with specific AlexaFluor488-labeled anti-mouse secondary antibody, 1:50 (Life Technologies) for 45 min at room temperature. Fluorescence was detected with a Leica SP5 confocal microscope and analyzed by ImageJ software.
3
Immunofluorescence Imaging of Transfected Cells
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Transfected cells on glass coverslips were washed twice with PBS and fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 in PBS, and blocked in 2% Bovine Serum Albumin in PBS for 10 min each at room temperature. Primary antibodies anti-HA.11 (BioLegend, 901514, San Diego, CA, USA) or anti-A3H were used at a dilution at 1:500 and 1:50, respectively, in 0.1% Triton X-100 in PBS and incubated with cells for 20 min at room temperature. Cells were washed five times with 0.1% Triton X-100 in PBS prior to incubation with Alexa Fluor 488-labeled anti-mouse secondary antibody (Thermo Fisher, A-11001) at a 1:1000 dilution in 0.1% Triton X-100 in PBS. Cells were again washed five times prior to mounting glass coverslips in ProLong Gold antifade reagent containing DAPI (Thermo Fisher, P36935). Images were obtained using a Nikon E800 microscope at 40× magnification.
4
Immunofluorescent Staining of Tight Junction Proteins
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The tight junction proteins occludin and ZO-1 were stained with corresponding antibodies. Briefly, phosphate buffer saline (PBS) was used to perfuse the sacrificed mice from each group. Whole-brain tissues were then harvested and sliced into 8 μm-thick sections. The sections were embedded using OCT compound. After rehydration and blocking for 1 h with 10% horse serum solution (Dako China), the sections were stained with primary antibodies against occludin (#91,131, Cell Signaling Technology, 1:50) and ZO-1 (#13,663, Cell Signaling Technology, 1:100) overnight at 4°C, followed by Alexa Fluor 488-labeled anti-mouse secondary antibody (#A32731, Thermo fisher scientific, 1:1000) for 1 h at room temperature in darkness. The slides were mounted with ProLong Antifade Mountant (Thermo Fisher Scientific, USA). A Zeiss fluorescence microscope was used to visualize the images.
5
Immunofluorescence Staining of Transfected Cells
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Cells were seeded on coverslips and transfected with plasmids or infected with PEDV. For transfection of HeLa cells, total 2 µg of individual plasmids were transfected for 24 h using Lipofectamine 2000 according to the manufacturer׳s instructions (Invitrogen). Cells were then either treated with poly(I:C) for 12 h or IFN-β for 40 min. Cells were fixed with 4% paraformaldehyde in PBS overnight at 4 °C and permeabilized using 0.1% Triton X-100 for 15 min at room temperature (RT). After blocking with 1% BSA in PBS at RT for 30 min, cells were incubated with a primary antibody in PBS for 1–3 h. Cells were then washed three times with PBS and incubated with Alexa Fluor 488-labeled anti-mouse secondary antibody, or Alexa Fluor 594-labeled anti-rabbit secondary antibody (Thermo Scientific) for 1 h at RT in the dark. Cells were incubated with DAPI for 5 min at RT for nuclear staining. After washing with PBS, cover slips were mounted on microscope slides using Fluoromount-G mounting medium (Southern Biotech, Birmingham, AL), and visualized by fluorescence microscopy (Nikon Eclipse TS100). Confocal microscopy was conducted as described elsewhere (Kannan et al., 2009 (link)).
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