Cells were cultured in poly-HEMA-coated dishes to aggregate for 24 hours. Afterward, cells were collected and spun onto Fisherbrand™ Superfrost™ Plus microscope slides (Thermo Fisher), and fixed with 4% paraformaldehyde for 10 min. Cells were permeabilized using 0.25% Triton X-100 in PBS, followed by blocking with 2% BSA in PBS for 1 hour. Caveolin 1, CD44 (N-terminal), and CD44-ICD primary antibodies were then incubated with cells overnight at 4 °C. Cells were then washed with PBS and incubated with Alexa Fluor™ 488 and Alexa Fluor™ 568-conjugated secondary antibodies (Thermo Fisher) for 1 hour, and finally, nuclei were counterstained with DAPI. The images were taken on a confocal microscope (Nikon Confocal, Melville, NY).
Alexa fluor 488 and alexa fluor 568 conjugated secondary antibodies
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies are fluorescent labeling reagents. They are designed to bind to primary antibodies and emit fluorescent signals at specific wavelengths for use in various detection and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fisherbrand superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Nanog, by Cell Signaling Technology (1 mentions)
Ssea 1, by Santa Cruz Biotechnology (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Mouse anti hsp27 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti sm22α antibody, by Abcam (1 mentions)
Mouse anti hsp27 monoclonal antibody, by Cell Signaling Technology (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
α tubulin dm1a, by Merck Group (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
Hrp conjugated antibodies, by Jackson ImmunoResearch (1 mentions)
Picrotoxin, by Merck Group (1 mentions)
Anti ndp52, by GeneTex (1 mentions)
Anti glur2, by Merck Group (1 mentions)
Mg132, by ChemScene (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Apotome 2, by Zeiss (1 mentions)
6 protocols using alexa fluor 488 and alexa fluor 568 conjugated secondary antibodies
1
Immunofluorescence Analysis of Caveolins and CD44
2
Immunofluorescence Staining of Pluripotent Stem Cells
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AP staining was performed for 30 min at room temperature in the dark using the Vector Red AP Substrate Kit I according to the manufacturer's protocol. The cells were incubated with substrate solution at room temperature until suitable staining developed and then photographed with a DFC300FX mounted digital camera (Leica, Solms, Germany). For immunofluorescence staining, mES or iPS cells were fixed in 4% paraformaldehyde in PBS for 10 min, washed twice with PBS, and blocked with 1% FBS in PBS for 30 min; all procedures were performed at room temperature. The fixed specimens were incubated with primary antibodies for 1 h, followed by incubation with secondary antibodies for 1 h. The Oct4 (1:100; Santa Cruz Biotechnology), Sox2 (1:100; Santa Cruz Biotechnology), Nanog (1:100; cell signaling), SSEA-1 (1:100; Santa Cruz Biotechnology), and Trib2 (1:100; Proteintech Group) primary antibodies were detected by Alexa Fluor 488 and Alexa Fluor 568 conjugated secondary antibodies (Thermo Fisher Scientific). The specimens were finally washed and mounted in Vectashield medium with 4′,6-diamidino-2-phenylindole for nuclei visualization. The stained sections were visualized using laser scanning confocal microscopy (FluoView FV1000, Olympus Corporation, Tokyo, Japan).
3
Immunohistochemical Analysis of Aortic HSP27
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Formalin-fixed, paraffin-embedded aortic sections were deparaffinized and rehydrated before antigen retrieval. The sections were incubated with mouse anti-HSP27 monoclonal antibody (1:50, Cell Signaling) at 4 °C overnight, and then incubated with peroxide-conjugated anti-mouse IgG secondary antibody and stained with 3, 3-diaminobenzidine using the Vectastain ABC kit (Vector Laboratories). For double immunofluorescence staining, the sections were incubated with mouse anti-HSP27 antibody (1:50, Cell Signaling) and rabbit anti-SM22α antibody (1:200, Abcam) at 4 °C overnight. Sections treated only with normal IgG were used as negative controls. Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies (Invitrogen) were used.
4
Immunofluorescence Staining of Focal Adhesions
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Phalloidin-TRITC, and monoclonal antibodies against vinculin (hVIN-1), HA (HA-7), c-myc (9E10) and α-tubulin (DM 1A) were from Sigma-Aldrich. Hamster anti-mouse CD29 (HMβ1-1), hamster anti-mouse CD61 (2C9.G2), hamster IgG isotype control and FITC goat anti-hamster IgG were from Biolegend, and HRP-conjugated rabbit anti-hamster IgG was from Abcam. Polyclonal antibody against FAK-pY397 was from Biosource International. Polyclonal antibodies against Src-pan, Src-pY418 and paxillin-p-Y118 were from Invitrogen. Monoclonal anti-paxillin (349), anti-phosphotyrosine (PY20), anti-PTP1B (15/PTP1B) and anti-FAK (77) were from BD Transduction Laboratories. Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies were from Invitrogen. HRP-conjugated antibodies were from Jackson Immunoresearch.
5
Pharmacological Inhibition of Autophagy and Proteasome
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The drugs used in this study were as follows: picrotoxin (Sigma), a GABA-receptor inhibitor; two different autophagy inhibitors, 3MA (3-methyladenine; Santa Cruz Biotechnology) and Bafilomycin (Bafilomycin A1; AdipoGen); and the proteasome inhibitor MG132 (Chemscene). The antibodies used were as follows: anti-GluR2 (Millipore), anti-LC3 (M152–3; MLB), A0024 (Daco), T22 (Millipore), Tau5 (Millipore), anti-pS396-tau (Invitrogen), anti-NDP52 (GeneTex), Alexa Fluor 488– and Alexa Fluor 568–conjugated secondary antibodies (Invitrogen), gold-conjugated (5 nm) secondary antibodies (British BioCell International), and horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories).
6
Immunofluorescence Staining of Kidney Tissue and Cells
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After deparaffinization and rehydration, kidney tissue sections were incubated with retrieval solutions and heated in a microwave for antigen retrieval. Nonspecific binding was blocked with serum-free blocking solution for 30 min at room temperature. After overnight incubation with primary antibodies at 4 °C, the tissue sections were labeled with Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies (1:1000, Invitrogen) in the dark for 1 h at room temperature. Nuclei were detected with 4,6-diamidino-2-phenylindole (DAPI; 1:1000, Thermo Fisher Scientific).
For immunofluorescence staining, cells grown on chamber slides were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized using 0.5% Triton X-100 for 10 min, and blocked with 3% BSA for 30 min at room temperature. After overnight incubation with primary antibodies in 3% BSA at 4 °C, the cells were rinsed with PBS and labeled with fluorescent secondary antibodies in the dark for 1 h at room temperature. Cells were washed twice with PBS and incubated with DAPI in PBS at room temperature for 5 min. Images were acquired using a Zeiss Apotome.2 (Carl Zeiss). Detailed information regarding the primary antibodies used for immunofluorescence staining is provided in Supplementary Table1 .
For immunofluorescence staining, cells grown on chamber slides were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized using 0.5% Triton X-100 for 10 min, and blocked with 3% BSA for 30 min at room temperature. After overnight incubation with primary antibodies in 3% BSA at 4 °C, the cells were rinsed with PBS and labeled with fluorescent secondary antibodies in the dark for 1 h at room temperature. Cells were washed twice with PBS and incubated with DAPI in PBS at room temperature for 5 min. Images were acquired using a Zeiss Apotome.2 (Carl Zeiss). Detailed information regarding the primary antibodies used for immunofluorescence staining is provided in Supplementary Table
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