Cells were seeded in duplicates into µ-Dish 35 mm (60 000 cells/ibidi dish). After 48 h, cells were washed with sterile phosphate-buffered saline (PBS, Thermo Fisher) before being fixed in 4% paraformaldehyde for 30 min (10 min at 4°C followed by 20 min at room temperature). Cells were washed and then permeabilized for 4 min in 0.2% Triton in PBS. Cells were washed again and incubated in blocking buffer (1% bovine serum albumin, and 2% normal goat serum, in PBS) for 1 h at room temperature before incubating with the primary antibody for 2 h. The following primary antibodies were used: Nestin (Abcam, ab22035, 1:100), Vimentin (Abcam, ab8069, 1:200), NG2 (Abcam, ab83178, 1:200), Ki67 (Abcam, ab16667, 1:200), Vinculin (FAK100, Sigma 1:200), and F-actin (FAK100, Sigma 1:500). Secondary antibodies were applied for 1 h (goat anti-mouse Alexa Fluor 488 preadsorbed, Abcam, 1:750 dilution and goat anti-rabbit Alex Fluor 594 preadsorbed, Abcam, 1:750). Nuclei were stained with DAPI (Roche).
Alex fluor 594
Manufactured by Abcam
Alex Fluor 594 is a red-fluorescent dye commonly used in fluorescence-based applications. It has an excitation maximum at 590 nm and an emission maximum at 617 nm.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Fak100, by Merck Group (2 mentions)
Ab16667, by Merck Group (2 mentions)
Ab22035, by Abcam (2 mentions)
Ab83178, by Abcam (2 mentions)
Ab8069, by Abcam (2 mentions)
Alexa fluor 488, by Abcam (2 mentions)
F actin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dapi solution, by Thermo Fisher Scientific (1 mentions)
Anti runx2, by Abcam (1 mentions)
Alex fluor 488, by Abcam (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
3 protocols using alex fluor 594
1
Immunostaining of Neural Stem Cells
2
Immunostaining of Foxc1 and Runx2
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Cells were fixed in 4% paraformaldehyde (PFA) for 15 min and then washed with PBS. After incubation in a 5% bovine serum albumin (BSA, Sigma, USA) containing 3‰ Triton X-100 for 1 h, cells were then incubated with the following primary antibodies dissolved in 5% BSA at 4°C overnight: anti-Foxc1 (1/200; CST, USA), anti-Runx2 (1/500; Abcam, USA). On the next day, cells were incubated with the following secondary antibodies for an hour: Alex Fluor 594 and Alex Fluor 488 (1/1000; Abcam). DAPI solution (Invitrogen, USA) was used for nuclear staining. Images were taken with a Canon fluorescence microscope (Japan).
3
Immunocytochemistry Characterization of Neural Cells
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Cells were seeded in duplicates into µ-Dish 35 mm (60,000 cells/ibidi dish). After 48 hours, cells were washed with sterile phosphate buffered saline (PBS, Thermo Fisher) before being fixed in 4% paraformaldehyde for 30 minutes (10 minutes at 4°C followed by 20 minutes at room temperature). Cells were washed and then permeabilized for 4 minutes in 0.2% Triton in PBS. Cells were washed again and incubated in blocking buffer (1% bovine serum albumin (BSA), and 2% normal goat serum (NGS), in PBS) for 1 hour at room temperature before incubating with the primary antibody for 2 hours. The following primary antibodies were used: Nestin (Abcam, ab22035, 1:100), Vimentin (Abcam, ab8069, 1:200), NG2 (Abcam, ab83178, 1:200), Ki67 (Abcam, ab16667, 1:200), Vinculin (FAK100, Sigma 1:200), F-actin (FAK100, Sigma 1:500). Secondary antibodies were applied for 1 hour (goat anti-mouse Alexa Fluor 488 pre-adsorbed abcam, 1:750 dilution and goat antirabbit Alex Fluor 594 pre-adsorbed abcam, 1:750). Nuclei were stained with DAPI (Roche)
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