Cells were cultured on poly-L-Lysine coverslips (Corning, USA) to 80% con uency, washed with PBS and xed with 4% formaldehyde for 20 min before permeabilization with 0.5% Triton-X100 for 2 min. For TurboID-START staining, an anti-BirA antibody was used to visualize the cells with anti-mouse conjugated FITC. As a positive control, an antibody (Abcam, USA) was used for staining of an endogenous PM marker, Na-K ATPase. If an antibody was not needed for staining, the cells were then incubated with Texas Red conjugated Streptavidin-(Thermo Fisher Sci, USA) for 1 hour at room temperature. Nuclei was stained with DAPI for 10 minutes at room temperature. Coverslips were mounted on glass slides with Prolong Diamond Antifade mounting media (Thermo Fisher Sci, USA). The cells were imaged on an Olympus CKx41 microscope equipped with a DP74 digital camera system.
Dp74 digital camera system
Manufactured by Olympus
The DP74 is a digital camera system designed for microscopy and imaging applications. It features a high-resolution sensor capable of capturing detailed images. The DP74 is a versatile tool for various scientific and research purposes, providing reliable image capture and data collection.
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2 protocols using dp74 digital camera system
1
Visualizing TurboID-START Localization
2
Dual Immunofluorescence Analysis of TPX2 and N-Myc in Cell Lines and NB Tissues
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For double immunofluorescence of cell lines and NB tissues, sections were incubated with primary antibodies against TPX2 (1:500) and N-Myc (1:200; cat. no. 51705; Cell signaling Technology, Inc.) overnight at 4°C. Alexa Fluor® 546 goat anti-mouse IgG (1:500; cat. no. A-11030) and Alexa Fluor® 488 goat anti-rabbit IgG (1:200; cat. no. A-11008) (both Invitrogen; Thermo Fisher Scientific, Inc.) were used as secondary antibodies for 1 h at room temperature. Nuclear counterstaining was performed using ProLong® Gold Antifade Reagent with DAPI (cat. no. 727434; Invitrogen; Thermo Fisher Scientific, Inc.). Confocal images were acquired using a BX53 inverted microscope with a DP74 digital camera system (Olympus). Further details are provided in Appendix 1 .
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