Hec50 cells were seeded on coverslips then fixed with 2% paraformaldehyde for 20 min. Coverslips were rinsed 3 times with 1ml PBS and incubated with 80% ice-cold methanol for 1 h, followed by permeabilization for 25–30 min with 0.2% Triton X-100. Cells were blocked with 3% BSA then incubated with specific antibodies at 4 °C overnight. Anti-MTDH (1:100, #14065), anti-phospho-histone H2AX (Ser139) (1:400, #9718) were from Cell Signaling Technology (Danvers, MA). Then, cells were incubated with Alexa Fluor 546-conjugated anti-rabbit secondary antibody (1:200, Cell Signaling Technology) at room temperature for 2 h; nuclei were stained using mounting solution with DAPI (Vector Laboratories). Visualization was performed on a Zeiss 710 confocal microscope.
Alexa fluor 546 conjugated anti rabbit secondary antibody
Manufactured by Cell Signaling Technology
The Alexa Fluor 546-conjugated anti-rabbit secondary antibody is a fluorescently labeled reagent used to detect and visualize rabbit primary antibodies in various applications such as immunofluorescence, flow cytometry, and Western blotting. The Alexa Fluor 546 dye provides a bright, photostable signal that can be detected using standard rhodamine filter sets.
Automatically generated - may contain errors
2 protocols using alexa fluor 546 conjugated anti rabbit secondary antibody
1
Immunofluorescence Staining of MTDH and γH2AX
2
Immunofluorescence Assay for LAMP2
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Coverslips were seeded with 1 × 105 PC3 cells for 24 h, then incubated with 250 ppm CO for 30 h. Coverslips were rinsed with PBS then fixed with 2% paraformaldehyde for 20 min, followed by permeabilization for 30 min with 0.2% Triton X‐100. Cells were blocked with 3% BSA and then incubated with anti‐LAMP2 (1:50, DSHB GAM‐568) at 4 °C overnight. Cells were then incubated with Alexa Fluor 546‐conjugated anti‐rabbit secondary antibody (1:200, Cell Signaling Technology) at room temperature for 2 h; nuclei were stained using mounting solution with DAPI (Vector Laboratories). Visualization was performed on a Zeiss 980 confocal microscope.
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