Human dermal fibroblasts or HaCaT cells were plated in 24-well culture plates. The cells were washed with PBS and fixed in 4% (wt/vol) PFA in PBS for 15 min at room temperature. Then the cells were washed with PBS and permeabilized with 0.1% (vol/vol) Triton X-100 in PBS for 15 min. After washing with PBS, the cells were incubated with 10% BSA in PBS for 30 min and then with antibodies specific for β-catenin (1:100; BD), CXXC5 (1:50; Santa Cruz Biotechnology, Inc.), keratin 14 (1:1,000; Covance), collagen I (1:500; Abcam), phalloidin (1:200; Molecular Probes), or α-SMA (1:200; Abcam) at 4°C overnight. The cells were washed in PBS and incubated with Alexa Fluor 488– or Alexa Fluor 555–conjugated IgG secondary antibody (1:400; Molecular Probes) at room temperature for 1 h. Cell nuclei were counterstained with DAPI for 10 min and the stained samples were examined under a LSM510 META microscope using 405-, 488-, or 543-nm laser excitation lines.
Alexa fluor 555 conjugated igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 555–conjugated IgG secondary antibody is a fluorescent-labeled antibody that binds to the Fc region of primary antibodies. It is used in immunoassay and imaging applications to detect and visualize target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
α sma, by Abcam (1 mentions)
Collagen 1, by Abcam (1 mentions)
β catenin, by BD (1 mentions)
Keratin 14, by Fortrea (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Anti pcna, by Santa Cruz Biotechnology (1 mentions)
Anti cd34, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti β catenin, by BD (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
Anti cxxc5, by Santa Cruz Biotechnology (1 mentions)
Anti cd68, by Abcam (1 mentions)
Anti keratin 14, by Fortrea (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Ab15580, by Abcam (1 mentions)
6 protocols using alexa fluor 555 conjugated igg secondary antibody
1
Immunofluorescent Staining of Cell Cultures
2
Immunofluorescence Analysis of Tissue Sections
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4-µm paraffin sections were deparaffinized and rehydrated. For antigen retrieval, the slides were autoclaved in 10 mM sodium citrate buffer. Sections were blocked in PBS containing 10% BSA at room temperature for 30 min. The sections were incubated overnight at 4°C with the following dilutions of primary antibodies: anti–β-catenin (1:100; BD), anti-CXXC5 (1:50; Santa Cruz Biotechnology, Inc.), anti–keratin 14 (1:1,000; Covance), anti–collagen I (1:500; Abcam), anti-PCNA (1:500; Santa Cruz Biotechnology, Inc.), anti-vimentin (1:250; Abcam), anti-CD34 (1:500; Abcam), anti-CD68 (1:200; Abcam), and anti–p-Erk (1:50; Cell signaling Technology). The slides were washed with PBS, incubated with Alexa Fluor 488– or Alexa Fluor 555–conjugated IgG secondary antibody (1:400; Molecular Probes) at room temperature for 1 h, and counterstained with DAPI (1:5,000; Boehringer Mannheim). The images were captured using a LSM510 META confocal microscope (Carl Zeiss) after excitation with 405-, 488-, or 543-nm laser lines.
3
MRGPRX2 Expression in Osthole-Treated LAD2 Cells
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LAD2 cells were treated with osthole (72 μM) at different timepoints (0, 15, and 30 min). After stimulation, cells were pelleted, washed with PBS, cytospun onto slides and fixed with 4% paraformaldehyde at room temperature. The slides were then permeabilized with 0.5% saponin for 10 min and blocked at room temperature for 1 h with blocking buffer (1% bovine serum albumin). After three washes with PBS with 0.1% Tween (PBST), slides were incubated with anti-human MRGPRX2 antibody (Novus Biologicals, Centennial, CO, United States) in dilution buffer (PBS containing 1% BSA, 1:500 dilution) in a humidified chamber at room temperature for 1 h. Slides were washed 3 times with PBST and incubated with anti-rabbit Alexa Fluor 555-conjugated IgG secondary antibody (Invitrogen) in dilution buffer (1:300) for 1 h at room temperature in the dark followed by incubation in 300 μM DAPI (1:1000 dilution). Slides were then washed 3 times in PBST and mounted using the ProLongTM Diamond antifade mounting media (Invitrogen). Confocal images were obtained with the Olympus FV 1000 confocal laser scanning microscope (Olympus America, Center Valley, PA, United States) and images were analyzed using the ImageJ software.
4
Immunofluorescence analysis of pancreatic islet cells
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Isolated islets were seeded in 24‐well plates and maintained in RPMI containing 10% FCS, 20 mM L‐glutamine, and 100 U/L penicillin/streptomycin with or without KY19334. For transient transfection, islets were transfected with siRNA using lipofectamine (Invitrogen) in Opti‐MEM (Gibco). Islet cells were fixed with 4% paraformaldehyde in PBS for 30 min at 20°C. The cells were then permeabilized and blocked with 0.5% Triton X‐100 and 5% BSA in PBS for 30 min at 20°C. Cells were then incubated overnight at 4°C with the following dilutions of primary antibodies: anti‐insulin (1:500, I2018, Sigma‐Aldrich) and anti‐Ki67 (1:100, ab15580, Abcam). Additionally, the cells were incubated with Alexa Fluor 488‐conjugated (1:300, A‐11001, Invitrogen) or Alexa Fluor 555‐conjugated IgG secondary antibody (1:300, A‐21428, Invitrogen) at 20°C for 1 h and counterstained with DAPI (1:5,000, D9564, Sigma‐Aldrich). The stained cells were captured using an LSM700 META confocal microscope (Carl Zeiss) after excitation with 405, 488 or 543 nm laser lines.
5
Immunofluorescence Staining of Cytoskeleton
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Cells grown on p-L-O and fibronectin-coated cover glasses were fixed in 4% PFA for 10 minutes, followed by permeabilization with 0.1% Triton X-100 for 15 minutes, blocking in 5% bovine serum albumin (BSA) for 1 hour, and primary antibody incubation overnight at 4°C. Primary antibodies were removed, and cells were washed with PBS and incubated for 1 hour at room temperature with either Alexa Fluor 488- or Alexa Fluor 555-conjugated IgG secondary antibodies (Invitrogen). Cell nuclei were counterstained by incubating the cells in 4′, 6-diamidino-2-phenylindole (DAPI; Sigma). For cytoskeleton staining, cells were fixed with 4% PFA, permeabilized, blocked, and incubated with Alexa Fluor 568 phalloidin (1:50) for 30 minutes. Immunofluorescent images were captured using fluorescent microscope (Nikon). Confocal images were obtained by LSM 510 META microscope (Carl Zeiss).
6
Immunofluorescence Staining of Cultured Cells
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Cells grown on gelatin-coated cover glasses were fixed in 4% PFA for 10 min, followed by permeabilization with 0.1% Triton X-100 for 15 min, blocking in 5% BSA for 1 h, and primary antibody incubation overnight at 4 °C. Primary antibodies were removed, and cells were washed with PBS and incubated for 1 h at room temperature with either Alexa Fluor 488- or Alexa Fluor 555-conjugated IgG secondary antibodies (Invitrogen). Cell nuclei were counterstained by incubating the cells in DAPI. Immunofluorescence images were captured using a confocal microscope (LSM510; Carl Zeiss).
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