Cells were cultured on a confocal dish and fixed with 4% paraformaldehyde (Sigma Aldrich) for 10 min. 0.1% Triton X-100 was used for permeabilization for 5 min. To inhibit nonspecific binding of antibodies, cells were incubated with 1% normal goat serum for 30 min. Next cells were incubated with 1:100 dilution of primary antibody for 2 h at room temperature and washed with PBS three times. After washing, cells were incubated with Alexa Fluor 488 or 555-conjugated secondary antibody (1:300) in dark for 1 h in room temperature. Stained images were visualized by a super-resolution radial fluctuations (SRRF) imaging system (Andor Technology, Belfast, UK). Relative Fluorescence intensity was analyzed by using ImageJ software.
Super resolution radial fluctuations imaging system
Manufactured by Oxford Instruments
Sourced in United Kingdom
The Super-resolution Radial Fluctuations (SRRF) imaging system is a microscopy technique that enables high-resolution imaging of biological samples. The core function of the SRRF imaging system is to analyze the temporal fluctuations of fluorescent signals within the sample to reconstruct a super-resolved image, providing greater spatial resolution than conventional fluorescence microscopy.
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4 protocols using super resolution radial fluctuations imaging system
1
Immunocytochemistry Protocol for SRRF Imaging
2
Immunocytochemistry of UCB-MSCs
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For immunocytochemistry, UCB-MSCs were fixed with 4% PFA for 10 min, then incubated in 0.5% Tween-20 for 10 min. Cells were incubated for 2 h with primary antibodies, in PBS containing 0.1% Tween-20 (PBST; 1:100 dilution), then washed three times with PBS. Cells were then incubated with Alexa Fluor 488 or 555-conjugated secondary antibodies in PBST (1:100 dilution) for 1 h. Immunofluorescence-stained samples were visualized using a super-resolution radial fluctuations (SRRF) imaging system (Andor Technology, Belfast, UK). The relative fluorescence intensity of HIF1α/DAPI was quantified using the ImageJ software.
3
Immunocytochemistry Visualization of HIF1α
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For the immunocytochemistry, UCB-MSCs were fixed with 4% paraformaldehyde (PFA; Lugen Sci, Seoul, Korea, #LGB-1175) for 10 min, and then incubated in 0.5% Tween-20 for 10 min. Cells were incubated with primary antibodies in PBS containing 0.1% Tween-20 (PBST; 1:100 dilution) for 2 h and washed with PBS three times. Cells were incubated with Alexa Fluor™ 488 or 555-conjugated secondary antibodies in PBST (1:100 dilution) for 1 h. Immunofluorescence stained samples were visualized by a super-resolution radial fluctuations (SRRF) imaging system (Andor Technology, Belfast, UK) [35 (link)]. Relative fluorescence intensities of HIF1α, HIF1α/BICD1 and HIF1α/Dynein IC were quantified with the ImageJ software. The co-localization rate of HIF1α with BICD1 was analyzed with the MetaMorph™ software (Universal Imaging, West Chester, PA, USA).
4
Quantitative Fluorescence Imaging of Neuronal Proteins
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Hippocampal neurons on a coverslip or SH-SY5Y cells on a confocal dish (Thermo Fisher) were fixed with 4% paraformaldehyde for 15 min and incubated in 0.3% Tween-20 for 5 min. Cells were placed in 5% normal goat serum (NGS) in PBS for 1 h and incubated with primary antibody dissolved in 5% NGS for overnight in 4 °C. Next, the cells were incubated for 2 h at room temperature with Alexa FluorTM secondary antibodies (Thermo Fisher, 1:100). Images were acquired by super-resolution radial fluctuations (SRRF) imaging system (Andor Technology, Belfast, UK). The fluorescent intensity analysis and colocalization analysis with Pearson’s correlation coefficient were acquired by Fiji 1.53 software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The values of Pearson’s correlation coefficient or the relative ratio of Pearson’s correlation coefficient (Compared to control) were shown in the quantification graph next to immunofluorescence images.
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