HuCCT1 cells were seeded and incubated on Chamber Slides (Lab-Tek II, Thermo Scientific, Waltham, MA, USA) for 24 h. The cells were fixed with 4% paraformaldehyde for 30 min and 100% methanol for 10 min. The slides were then blocked in 4% bovine serum albumin (Millipore, Biillerica, MA, USA) for 30 min and incubated for 48 h at 4°C with the appropriate combinations of mouse, rabbit and goat antibodies diluted 1:100 (STMN1 rabbit antibody; Cell Signaling Technology, Danvers, MA, USA; and p27 mouse antibody; Santa Cruz Biotechnology) in antibody dilution solution (Olink Bioscience, Uppsala, Sweden). After washing, the slides were incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes (Olink Bioscience) and a proximity ligation was performed using the Duolink Detection Reagent Kit (Olink Bioscience) according to the manufacturer's protocol. Nuclei were stained with Duolink In Situ Mounting Medium with DAPI (Olink Bioscience). Images were acquired with an All-in-one Fluorescence Microscope (Keyence Corporation, Osaka, Japan).
Duolink in situ mounting medium with dapi
Manufactured by Olink
Sourced in Sweden
Duolink In Situ Mounting Medium with DAPI is a lab equipment product used for mounting and preserving samples for microscopic analysis. It contains the dye DAPI, which stains DNA and allows for visualization of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Duolink detection reagent kit, by Olink (1 mentions)
All in one fluorescence microscope, by Keyence (1 mentions)
Duolink pla rabbit minus and pla mouse plus proximity probes, by Olink (1 mentions)
Spe confocal system, by Leica camera (1 mentions)
2 protocols using duolink in situ mounting medium with dapi
1
Proximity Ligation Assay for STMN1-p27 Interaction
2
Multiplexed Protein Interaction Imaging
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Cells were fixed with 4% formaldehyde in PBS for 20 min, permeabilized with cold (−20 °C) 70% ethanol for 20 min, blocked with 8% BSA in PBS for 1 h, incubated with the first antibody (caspase-8, 1/50 dilution; FADD, 1/100 dilution; NOX2, 1/250 dilution; p47PHOX, 1/250 dilution) in 1% BSA in PBS for 1.5 h, then with PLUS and MINUS PLA probes (Olink Bioscience, Uppsala, Sweden) for 1 h at 37 °C, then with ligation mix (Olink Bioscience) for 30 min at 37 °C, then with amplification mix (Olink Bioscience) for 100 min at 37 °C before being mounted using Duolink In Situ Mounting Medium with DAPI (Olink Bioscience). Confocal images were acquired on a Leica SPE Confocal system, sequential acquisition using 63X/1.3 oil immersion. 3D pictures were realized with the Bitplane Imaris software v. 7.7.2.
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