Super-resolution images were obtained on a commercial Nikon N-STORM super-resolution microscope. Raw images were processed with the ImageJ plugin ThunderStorm to obtain the final super-resolution images (25 (link)). See supporting information for additional details.
N storm super resolution microscope
Manufactured by Nikon
Sourced in Japan
The N-STORM super-resolution microscope is a high-resolution imaging system designed for advanced microscopy applications. It utilizes structured illumination microscopy (SIM) technology to achieve a resolution beyond the diffraction limit of traditional optical microscopes. The N-STORM system enables the visualization and study of cellular structures and processes at the nanoscale level.
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Lab products found in correlation
Alexa647 conjugated azide, by Thermo Fisher Scientific (1 mentions)
Dmi6000b, by Leica (1 mentions)
Las af lite, by Leica (1 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Airyscan 2, by Zeiss (1 mentions)
Slowfade diamond antifade reagent, by Thermo Fisher Scientific (1 mentions)
Cover glasses, by Matsunami (1 mentions)
A1 confocal laser microscope, by Nikon (1 mentions)
Glass bottomed dishes, by Iwaki (1 mentions)
4 protocols using n storm super resolution microscope
1
Super-Resolution Microscopy Protocol
2
Doxycycline-Induced PAR1 Nanoscale Visualization
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A549 cells were seeded in 35-mm N-STORM super-resolution microscope dishes and were grown to 60% confluency. Next, 0.2 μM doxy-yne was added to the cells and incubated for 4 h at 37°C in the presence of 5% CO2, followed by UV irradiation (365 nm) for 30 min. The cells were then fixed and permeabilized and 0.1 mM TBTA, 1 mM TCEP, 0.1 mM Alexa 647-conjugated azide (Thermo Fisher, USA) and 1 mM CuSO4 were added to the cells for 2 h for the click reaction. After the click reaction, the cells were incubated with rabbit polyclonal anti-PAR1 (CST, USA) in a humidified chamber overnight at 4°C and then were incubated with Cy3B-conjugated goat anti-rabbit secondary antibody for 2 h at room temperature, followed by seal slicing. The N-STORM super-resolution microscope (Nikon, Japan) was used to measure doxycycline and PAR1 co-localization on the nanoscale level.
3
Quantifying Tight Junction Protein Expression
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The expressions of claudin-5, occluding, and ZO-1 were detected by IHC analysis. Briefly, the rat brain was fixed in 4% paraformaldehyde for 20 min at 37 °C. Rat brain sections were incubated with claudin-5 (1:400), occludin (1:700), and ZO-1 (1:500) primary antibodies at 4 °C overnight. Subsequently, the sections were incubated with HRP-conjugated anti-rabbit/goat IgG at 37 °C for 20 min and color was then rendered by the diaminobenzidine (DAB) Immunohistochemistry Color Development Kit. IHC Images were visualized using Leica DMI6000B fluorescence microscopy with LAS AF LITE image processing software (Leica, Weztlar, Germany). HBMECs were stained with phalloidin (1:200, Yeasen Biotech Company, Shanghai, China) and DAPI to show F-actin and cell nucleus, respectively [75 (link)]. Finally, the images were gained using an N-STORM super-resolution microscope (Nikon, Tokyo, Japan).
4
Immunofluorescence Imaging of HeLa Cells
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First, we grew HeLa cells on glass-bottomed dishes (#3911–035; Iwaki, Chiba, Japan), cover glasses (Matsunami, Osaka, Japan), or a glass-bottomed 8-well cell chamber (0030 742.036; Eppendorf, Hamburg, Germany). We washed the cells twice with PBS, fixed them with 3.6% PFA in PBS for 20 min, permeabilized them with 0.2% Triton X-100 for 3 min, and blocked them with 1% BSA and 0.02% Triton X-100 in PBS for 1 h each at room temperature. Next, the cells were probed with the respective primary antibodies overnight at 4 °C and incubated with the respective secondary antibodies for 1 h at room temperature. Antibody information is available in Supplementary Table S1 . The cells were washed thrice with PBS, and coverslips were mounted with SlowFade Gold antifade reagent (Invitrogen) or SlowFade Diamond antifade reagent (Invitrogen). For N-STORM analysis, we filled the cell chambers with the STORM imaging buffer with MEA prepared according to the manufacturer’s instructions. Next, we obtained images using an A1 + confocal laser microscope (Nikon), an N-STORM super-resolution microscope (Nikon), or an LSM 980 with Airyscan 2 (ZEISS, Oberkochen, Germany).
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