After five days in culture, 1 ml of room temperature PBS containing 8% (w/v) paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) was added to the culture solution for a final concentration of 4% paraformaldehyde. After 30 min, the paraformaldehyde was removed and washed three times with PBS before a PBS blocking solution containing 10% fetal bovine serum (FBS, Invitrogen, Life Technologies, Carlsbad, CA) and 0.1% Triton X-100 was added to the cells and incubated at 4°C. After 12 h, the blocking solution was removed and a PBS solution containing 2% of FBS, 0.1% Triton X-100, and RT-97 antibody to label neurofilament (1:100, Developmental Studies Hybridoma Bank, Iowa City, IA) was added to the DRG cultures. After 8 h, the solution was removed, and the DRG were washed three times with PBS. Then, a PBS solution containing 2% of FBS, 0.1% Triton, and donkey anti-mouse antibody conjugated with Alexa-594 (1:1000 dilution, Sigma-Aldrich, St. Louis, MO) was added to the DRG cultures and incubated for 1 h. Finally, a solution of DAPI (1:1000, Sigma-Aldrich, St. Louis, MO) was added for 15 min before washing the cells three times with PBS. The cells were imaged with an Olympus DSU spinning disc confocal microscope (Center Valley, PA).
Fetal bovine serum (fbs)
Manufactured by Developmental Studies Hybridoma Bank
Fetal Bovine Serum (FBS) is a commonly used cell culture supplement derived from the blood of fetal bovine. It provides growth factors, vitamins, and other essential nutrients to support the growth and proliferation of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Dsu spinning disc confocal microscope, by Olympus (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Yol1 34, by Bio-Rad (1 mentions)
Schneider s insect medium, by Thermo Fisher Scientific (1 mentions)
Autoquant x, by Media Cybernetics (1 mentions)
Concanavalin a coated, by Merck Group (1 mentions)
Alexa fluor 568 and 647, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Adobe (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Orca r2, by Hamamatsu Photonics (1 mentions)
2 protocols using fetal bovine serum (fbs)
1
Immunostaining of Cultured Dorsal Root Ganglia
2
Imaging Cellular Structures in S2 Cells
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S2 cells transiently expressing CD8-GFP were grown on concanavalin A–coated (0.5 mg/ml; EMD Millipore) glass coverslips at 25°C in Schneider’s insect medium (Gibco) + 10% FBS (Invitrogen) and fixed with 4% paraformaldehyde for 10 min and subsequently extracted with 0.1% Triton X-100 for 10 min. After short washes in PBS and blocking with 10% FBS, cells were incubated with mouse anti-lamin Dm0 (clone ADL67, 1:50; Developmental Studies Hybridoma Bank) and rat anti–α-tubulin (YOL1/34, 1:100; Serotec), followed by short washes in PBS and incubation with Alexa Fluor 568 and 647 (1:1,000; Invitrogen). DNA was counterstained with DAPI (1 µg/ml; Sigma-Aldrich) before coverslips were mounted in 90% glycerol + 10% Tris, pH 8.5, + 0.5% N-propylgallate on glass slides. Images were acquired on an AxioImager Z1 (100×, Plan Apochromatic oil DIC objective lens, 1.4 NA; all from Carl Zeiss) equipped with a charge-coupled device (CCD) camera (ORCA-R2; Hamamatsu Photonics) using the Zen software (Carl Zeiss) and blind deconvolved using Autoquant X (Media Cybernetics). Images were processed (contrast adjustments) in Adobe Photoshop CS4 (Adobe) and represent either maximum intensity projections of deconvolved z stacks (step size: 0.22 µm; DAPI; α-tubulin) or a single slice (CD8-GFP; lamin Dm0).
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