HEK293 cells were grown on glass coverslips. After 10% formalin fixation (10 min), coverslips were washed 3x10 min in phosphate buffered saline (1x PBS: 137 mM NaCl, 2.7 mM KCl, 18 mM KH2PO4, 10 mM Na2HPO4) and cells were subsequently permeabilized with a 1x PBS, 0.3% Triton-X100 solution. Following blocking with a 1% BSA, 0.2% gelatin, 0.05% saponin in 1x PBS solution and washing with a 0.1% BSA, 0.2% gelatin, 0.05% saponin in 1x PBS solution, fixed cells were treated overnight at 4°C with a mouse anti-MycTag (9E10 epitope) antibody diluted in an adequate buffer (0.1% BSA, 0.1% sodium azide, 0.3% triton X-100 in 1x PBS). Then, coverslips were rinsed 3 times in washing solution. Cells were further incubated with a fluorescent secondary anti-mouse-Alexa647 antibody (1:500, ThermoFischer, cat. no. A32728) to detect antigen-antibody complexes and counter-stained with DAPI (500 ng/ml, Sigma Aldrich, cat. no. 32670-25MG). Slides were scanned with a Pannoramic Midi II scanner (3DHISTECH Ltd.).
Anti mouse alexa647 antibody
Manufactured by Thermo Fisher Scientific
The Anti-mouse-Alexa647 antibody is a fluorescently labeled secondary antibody used for immunofluorescence detection. It binds to primary antibodies raised in mouse and is conjugated to the Alexa Fluor 647 dye, which can be detected using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
2 protocols using anti mouse alexa647 antibody
1
Immunofluorescent Staining of MycTag in HEK293 Cells
2
Integrin and Actin Cytoskeleton Analysis in Lymphoid Cells
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Flow cytometry was performed on peripheral blood mononu-clear cells, LCL, and cultured primary B cells using an LSRFortessa X-20 (BD Biosciences). The results were processed using FlowJo v10 software (TreeStar Inc., St. Ashland, OR, USA). To determine integrin expression at the cell surface, LCL were labeled with an anti-human CD11a antibody (TS2/4; Biolegend) for total CD11a expression, or an anti-human CD11a antibody (Hl111; Biolegend) for inactive/closed conformation-CD11a expression, or an anti-human CD54 antibody (Biolegend), followed by an anti-mouse-Alexa647 antibody (ThermoFisher Scientific). To determine F- and G-actin content in two LCL samples side by side, one sample was incubated with an anti-human CD54 antibody (Biolegend) for 30 min on ice and thereafter labeled with DNaseI-Alexa488 (ThermoFisher Scientific) and phalloidin-Alexa568 (ThermoFisher Scientific).
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