Cells were cultured in conditional media containing 0.8 or 0 mM serine with an addition of 10% FBS on glass coverslips (VWR) in six-well dishes for 24 hr. Next, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After one wash in 1× PBS, cells were permeabilized in 0.15% Triton X-100 for 15 min at room temperature. Cells were then blocked in 10% BSA for 20 min at room temperature, followed by incubation in primary antibody solution (1:500 dilution in 10% BSA) for overnight at 4°C. After three washes using 1× PBS, cells were incubated in secondary antibody solution in 10% BSA and followed by another three times of wash. Cells were then mounted onto glass slides using Fluoromount-G mounting solution (Southern Biotech) for imaging acquisition on a spinning-disk confocal microscope (Leica) using a × 100/1.4NA oil or a ×40/1.25NA oil (Leica Plan Apochromat) objective. Images were acquired and processed using the Leica LAS AF program software.
Las af program software
Manufactured by Leica
The LAS AF (Leica Application Suite Advanced Fluorescence) program software is a comprehensive imaging and analysis platform designed for microscopy. It provides tools for image acquisition, processing, and analysis to support a wide range of fluorescence microscopy applications.
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2 protocols using las af program software
1
Immunofluorescence Imaging of Serine Metabolism
2
Immunofluorescence Imaging of Serine Metabolism
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Cells were cultured in conditional media containing 0.8 or 0 mM serine with an addition of 10% FBS on glass coverslips (VWR) in six-well dishes for 24 hr. Next, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After one wash in 1× PBS, cells were permeabilized in 0.15% Triton X-100 for 15 min at room temperature. Cells were then blocked in 10% BSA for 20 min at room temperature, followed by incubation in primary antibody solution (1:500 dilution in 10% BSA) for overnight at 4°C. After three washes using 1× PBS, cells were incubated in secondary antibody solution in 10% BSA and followed by another three times of wash. Cells were then mounted onto glass slides using Fluoromount-G mounting solution (Southern Biotech) for imaging acquisition on a spinning-disk confocal microscope (Leica) using a × 100/1.4NA oil or a ×40/1.25NA oil (Leica Plan Apochromat) objective. Images were acquired and processed using the Leica LAS AF program software.
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