To identify in situ COA7 and TOM20 localization, 5 × 103 or 1 × 104 cells were seeded in glass-bottom slides (ibidi, 80427) and grown overnight. The next day, cells were treated with 1 or 2 μM of the analogs or DMSO as the control. After 48 h, cells were fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were then washed (0.2% Triton X-100 in PBS with Ca2+ and Mg2+) and blocked in 3% BSA (in wash buffer) for 1 h. Cells were incubated with primary antibody overnight at 4°C. Primary antibodies used were TOM20 (Proteintech, 66777-1-Ig) and COA7 (Proteintech, 25361-1-AP). The next day, cells were washed and incubated with secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI) (Sigma) for 50 min at room temperature. Washed cells were then imaged using a Leica SP8 confocal microscope with a 63× objective. At least 60 cells were imaged per treatment and per three biological replicates. Images were analyzed, processed, and the Pearson’s correlation coefficient determined using ImageJ/FIJI.73 (link)
Glass bottom slides
Manufactured by Ibidi
Glass-bottom slides are laboratory equipment designed to facilitate microscopic observation and analysis. They feature a transparent glass surface that allows for the placement and examination of samples under a microscope.
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Lab products found in correlation
2 protocols using glass bottom slides
1
Visualizing COA7 and TOM20 Localization
2
Visualizing Disordered Protein Assemblies
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For phase-separation experiments, soluble GA and PA DPRs (stock concentration = 100 μM) were diluted in ddH2O to either 20, 10 or 1 μM. After vortexing, the mixture was pipetted onto glass-bottom slides (Ibidi), and assembly formation was monitored over time. Images were acquired immediately, as well as after 2 and 24 h using a Leica SP5 confocal microscope with a 63× 1.4 NA oil immersion objective, 561 channel. To capture fine details for 3D rendering, assemblies were imaged with a z-stack following Nyquist sampling for optimized pixel density. Huygens Professional version 19.10 (Scientific Volume Imaging, http://svi.nl ) was used to deconvolve z-stack data using the CMLE algorithm (with SNR:10 and 40 iterations) and subsequently for 3D-volume and surface rendering thus generating Videos 1 and 2 . The 3D rendering for Videos 3 and 4 was performed using the software Imaris v7.7.2 (Bitplane); no deconvolution was applied here. Number, circularity and size of DPR assemblies were quantified with FIJI (Schindelin et al, 2012 (link)) and plotted using GraphPad Prism 8. The FIJI plug-in Trainable Weka Segmentation (Arganda-Carreras et al, 2017 (link)) was used to finely measure the circularity of liquid droplets in heterogeneous PA assemblies (20 μM, 24 h) (Fig S3D and E ).
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