N2a cells (3 × 104 cells/well) were plated in 8-well chamber slides and grown overnight to 80% confluence. Cells were treated with 2.5 μg/ml eCIRP for 16 h followed by three Phosphate-Buffered Saline (PBS) washes and the membrane stained with Biotium MemBrite™ Fix 488/515 using the staining kit. Next, cells were washed, fixed for 15 min by 4% paraformaldehyde, permeabilized for 10 min with PBS containing 0.1% Triton X-100 and blocked with 1% BSA, 100 mM glycine in PBS for 30 min at RT. The slides were incubated overnight at 4°C with 1:100 diluted calpain-1 antibody, washed and incubated for 1 h at RT with donkey anti-rabbit IgG-Alexa Fluor 594 and Hoechst 33342. After washing three times with PBS, slides were mounted immediately on Vectashield mounting medium (Vector Laboratories). The images were acquired using a Zeiss Axio Observer equipped with LSM880 confocal microscopy system (Zeiss). The images obtained by confocal microscopy were merged and combined using FIJI ImageJ.
Lsm 880 confocal microscopy system
Manufactured by Zeiss
Sourced in Germany
The LSM 880 is a confocal microscopy system designed by Zeiss. It is a high-performance imaging platform that utilizes laser scanning technology to capture detailed, high-resolution images of microscopic samples.
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3 protocols using lsm 880 confocal microscopy system
1
Visualizing Calpain-1 Localization in N2a Cells
2
Immunofluorescence Staining of IGFBP3 and Orai
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HCAECs were seeded on cover glass at an appropriate density and cultured in an incubator. After being washed with PBS, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 and 3% BSA (Bovine Serum Albumin) at room temperature. They were blocked for 30 min, washed with PBS, and incubated with anti-IGFBP3 or Orai1–3 primary antibodies overnight at 4°C in a humid chamber. An FITC (fluorescein isothiocyanate)-conjugated secondary antibody (diluted 1:100; Invitrogen) was incubated with the cells for 2 hours at room temperature. The nucleus of the cells was labeled with DAPI (4',6-diamidino-2-phenylindole). The immunofluorescence signals were detected and captured using an LSM 880 confocal microscopy system (Zeiss, Germany).
3
Immunofluorescence Visualization of Orai, VE-cadherin, and p-VE-cadherin
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MAECs (4×104 cells) were seeded on cover glass. After treatment, the cells were washed three times with PBS and fixed using 4% paraformaldehyde (Ebiogo, Hefei, China). Cell membranes were permeabilized with 0.2% Triton X-100 (Beyotime Biotechnology, China) for 15 min and blocked with 3% bovine serum albumin (Beyotime Biotechnology) for 2 hours at room temperature. Primary rabbit anti-Orai1–3, anti-VE-cadherin or anti-p-VE-cadherin Y731 antibodies (diluted 1:200) were incubated with the cells overnight. Then Orai1–3, VE-cadherin and p-VE-cadherin Y731 were labeled using FITC-conjugated secondary antibodies (diluted 1:100; Invitrogen) via incubation for 2 hours. The nucleus of each cell was labeled with 4',6-diamidino-2-phenylindole (DAPI). The immunofluorescence signals were detected and images were captured using an LSM 880 confocal microscopy system (Zeiss, Germany).
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