Cells grown on coverslips were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 10 min, and blocked in 3% normal donkey serum in PBS for 2 h at room temperature. Primary antibodies were diluted in 3% normal donkey serum in PBS and applied at 4 °C overnight. The primary antibodies used in these experiments were as follow: Nestin (Abcam; 1:3000), Sox2 (Santa Cruz; 1:500), after rinsing in PBS three times and incubating for 2 h with CF488 and CF543 (Biotium; 1:1000), coverslips were washed three times, cell nuclei were stained with DAPI. Images were acquired on a Leica TCS SP2 confocal fluorescence microscope.
Tcs sp2 confocal fluorescence microscope
Manufactured by Leica
The TCS SP2 is a confocal fluorescence microscope designed for high-resolution imaging. It is capable of capturing detailed images of fluorescently labeled samples. The system utilizes a laser-scanning approach to achieve optical sectioning and three-dimensional reconstruction of samples.
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Lab products found in correlation
Nestin, by Abcam (2 mentions)
Cf488, by Biotium (2 mentions)
Cf543, by Biotium (2 mentions)
Triton x 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Gbd 35 20, by NEST Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Tissue tek optimal cutting temperature compound, by Avantor (1 mentions)
Spotdetector bioapplication, by Thermo Fisher Scientific (1 mentions)
6 protocols using tcs sp2 confocal fluorescence microscope
1
Immunofluorescence Staining of Neural Stem Cells
2
Immunofluorescence Staining of Cultured Cells
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Cells were grown in 35 mm glass-bottom petri dishes (NEST, GBD-35-20). The indicated interfering RNA was transfected at various time points. Cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and washed with 0.2% Triton X-100 (Sigma-Aldrich, T8787) in PBS for 10 min. The cells were blocked in PBS containing 5% bovine serum albumin (BSA; Beyotime, ST023) for 30 min. Next, the cells were stained with primary antibodies and appropriate secondary antibodies for 1 h at 37°C. Wherever indicated, nuclei were stained with DAPI (Beyotime). Fluorescence was visualized with a TCS SP2 confocal fluorescence microscope (Leica).
3
Nestin Expression in Mouse Brain
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Mice were anesthetized and cardiac perfused through the heart with 4% paraformaldehyde. Brains were dissected out, post-fixed and stored in 30% sucrose. Brains were frozen in Tissue-Tek optimal cutting temperature compound (O.C.T.) (VWR, Richmond, IL) and coronally sectioned at a 25 μm interval. Tissue sections were blocked with 3% normal donkey serum (NDS, Jackson) in PBS for 30 minutes and then incubated with Nestin (Abcam; 1:3000) at 4 °C overnight followed by CF488 (Biotium; 1:1000) for 1 hour. DAPI was used as counter staining. Images were acquired and analyzed on a Leica TCS SP2 confocal fluorescence microscope.
4
Quantifying Autophagy Flux by LC3 Puncta
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Cells were fixed with 4% paraformaldehyde for 30 min, permeabilised with 0.2% Triton X-100 (Sigma-Aldrich, T8787) for 10 min, and blocked in PBS containing 5% bovine serum albumin (BSA; Beyotime, ST023) for 30 min. Cells were incubated with anti-LC3 (1:100) at 37°C for 1 h, followed by secondary antibodies conjugated to Alexa Fluor 488. The fluorescence signals were visualized with a TCS SP2 confocal fluorescence microscope (Leica). The Spot Detector BioApplication (Thermo Fisher Scientific) was used to acquire and analyze the images after optimization. Images of 500–1,000 cells for each treatment group were analyzed to obtain the mean LC3 fluorescence punctae number per cell (Tang et al., 2010 (link)). (n = 5 each group).
5
Immunocytochemistry of γH2AX
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Cells grown on coverslips were fixed in 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100 (Sigma), and blocked in 3% normal donkey serum in PBS for 30 minutes at room temperature. Rabbit anti-human γH2AX (Abcam; 1:1000) antibody was diluted in 3% normal donkey serum in PBS and applied at 4°C overnight. After rinsing with PBS three times and incubating for one hour with donkey anti-rabbit secondary antibody (CF543, Biotium; 1:1000), slides were washed three times in PBS and cell nuclei were stained with DAPI (Invitrogen) for 10 minutes at room temperature. Images were acquired on a Leica TCS SP2 confocal fluorescence microscope.
6
Immunofluorescence Imaging of Fixed Cells
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Cells were fixed in 4% paraformaldehyde, then rinsed with phosphate-buffered saline (PBS) three times. Cells were blocked in 3% normal donkey serum in PBS for 1 h. Cells then were incubated with primary antibody diluted in 3% normal donkey serum in PBS at 4 °C overnight. After that, cells were rinsed with PBS containing 0.1% Triton X-100 (Sigma) three times and incubated with secondary antibody for 1 h. Cell nucleus were stained with 4′,6-diamidino-2-phenylindole (Invitrogen) for 10 min and slides were washed three times in PBS containing 0.1% Triton X-100 (Sigma). Images were obtained on a Leica TCS SP2 confocal fluorescence microscope.
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