11 non-curved FSCMs, that were cut in halves, for CK3/12 (1:100; ab68260; Abcam, Cambridge, UK), Ecadherin (1:400; ab53033), integrin α6 (1:1000; ab20142), laminin-332 (1:25; ab11575; Abcam; Cambridge, UK), and additionally with ∆N P63 (1:300; sc-8609; Santa Cruz Biotechnology; CA, USA), a determinant for stemness. Corneal stromal cells were also cultured on the smooth side of FSCMs (P12261201), coated with and without rat tail collagen type I. The cells were stained for whole mount immunofluorescence with antibodies against the transmembrane receptors integrin-α6 (1:1000; ab20142) and integrin-β1 (1:50; ab52971; Abcam, Cambridge, UK), involved in cell-matrix adhesion, and counterstained for F-actin (Alexa Fluor 488 Phalloidin; A12379) to depict the cytoskeleton, and with DAPI for cell nuclei.
Ab20142
Manufactured by Abcam
Sourced in United Kingdom
Ab20142 is a laboratory equipment product offered by Abcam. It serves as a core function for various applications in research and scientific experiments. The detailed technical specifications and intended use of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab11575, by Abcam (1 mentions)
Ab68260, by Abcam (1 mentions)
Ab52971, by Abcam (1 mentions)
Ab53033, by Abcam (1 mentions)
Sc 8609, by Santa Cruz Biotechnology (1 mentions)
Fitc labeled secondary antibodies, by Bio-Synthesis (1 mentions)
Mab1950z, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Ix81 fluorescence microscope, by Olympus (1 mentions)
Tcs sp5 2 confocal microscope, by Leica (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab133682, by Abcam (1 mentions)
Sa00013 1, by Proteintech (1 mentions)
4 protocols using ab20142
1
Corneal Stromal Cell Adhesion on FSCMs
2
Immunostaining of Integrin α6 in Cells
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Cell suspensions were incubated on poly-L-lysine-coated slides to adhere and fixed for 10 min in 4% paraformaldehyde. After 1 h of blocking in 10% bovine serum albumin (BSA) in PBS, antibodies against Integrinα6 (ab20142, Abcam, Shanghai, China) were added at a 1:200 dilution in 10% BSA in PBS and incubated overnight at 4°C. After washing three times with 0.05% Tween-20 in PBS, FITC-labeled secondary antibodies (Biosynthesis Biotechnology Co., Ltd.) were added at a 1:100 dilution for 2 h. After washing again with 10% BSA-PBS, the cells were observed under an inverted fluorescence microscope.
3
Cell Surface Antigen Profiling by Flow Cytometry
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Cells were detached with PBS/EDTA (0.05%), pelleted, suspended in DPH + 10% FBS, and kept in suspension on a shaker for 1 h at 37°C for recovery. A total of 2 × 105 cells per well were pelleted in a v-bottom 96-well plate and washed with cold PBS + 2% FBS. Antibodies against CAR (clone RmcB, Merck, #05-644), HS (clone F58-10E4, amsbio, #370255S), alpha2 (clone P1E6, Merck, MAB1950Z), alpha3 (clone ASC-1, Merck, MAB2056), alphaV (clone P2W7, Abcam, ab11470), and alpha6 (clone MP4F10, Abcam, ab20142) were diluted in cold PBS + 2% FBS to a working concentration of 0.5–1 µg/mL. Cells were incubated with 100 µL antibody dilution for 30 min on ice in shaking conditions. Cells were pelleted, resuspended in PBS, pelleted again, and incubated with 100 µL secondary antibody dilution (goat anti-mouse IgG, IgM Alexa Fluor 488, Thermo Fisher Scientific, #A-10680) at a working concentration of 1 µg/mL in PBS + 2% FBS for 30 min on ice in shaking conditions. Cells were washed again and suspended in PBS + 2% FBS as above. Cell surface staining was measured by flow cytometry in a Bio-Rad ZE5 and analyzed with FlowJo version 10.
4
Immunofluorescence Assay of Epithelial-Mesenchymal Transition Markers
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Cells were seeded in 6-well plates containing three cover slides at a density of 5000 cells per well and cultured overnight. The cells were then fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 (Sigma, USA) for 5 min and blocked with 2% BSA (Sigma) for 1 h at room temperature. Subsequently, the cells were incubated with primary antibodies against E-cadherin (1:100, Proteintech, 20874-1-AP, USA), N-cadherin (1:100, Proteintech, 22018-1-AP), Vimentin (1:100, Proteintech, 10366-1-AP), integrin α6 (1:1000, Abcam, ab20142, UK) and integrin β4 (1:500, Abcam, ab133682) separately overnight at 4 °C. The next day, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody (1:100, Proteintech, SA00013-1/ SA00013-2) or Alexa Fluor 594-conjugated secondary antibody (1:100, Proteintech, SA00013-3/SA00013-4) for 1 h at room temperature in the dark. Cell nuclei were counterstained with DAPI (Sigma) for 5 min at room temperature in the dark. IF signals were visualized using an Olympus IX81 fluorescence microscope (Olympus) and a Leica TCS SP5II confocal microscope (Leica, Germany). Data were analyzed using ImageJ software.
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