HeLa cells were plated on coverslips and harvested at 48 h posttransfection. After being washed twice with phosphate-buffered saline (PBS), cells were fixed with 4% paraformaldehyde diluted by PBS and permeabilized using 0.1% Triton X-100. Then fixed cells were washed five times with PBS and incubated in blocking solution for 2 h. After washing off the blocking solution, cells were incubated with anti-HA (1:200; cat. no. H6908, Sigma) and anti-Flag (1:100; cat. no. F1804, Sigma) diluted in PBS containing 0.1% bovine serum albumin (BSA) for 2 h. Cells were washed three times with PBST and incubated with fluorescein (FITC) goat anti-mouse IgG (1:500; cat. no. 115-095-003, Jackson ImmunoResearch) and rhodamine (TRITC) goat anti-rabbit IgG (1:500; cat. no. 111-025-003, Jackson ImmunoResearch) for 1 h. Following four washes with PBS, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 1:2,000), and mounted on slides with Antifade Mounting Medium (Beyotime) for confocal microscopy analysis. The images were captured by Leica TCS SP5 confocal system.
Rhodamine tritc goat anti rabbit igg
Manufactured by Jackson ImmunoResearch
Rhodamine (TRITC) goat anti-rabbit IgG is a secondary antibody conjugate. It is used to detect and visualize rabbit immunoglobulin G (IgG) in various immunological techniques.
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3 protocols using rhodamine tritc goat anti rabbit igg
1
Immunofluorescence Staining of Transfected HeLa Cells
2
Immunofluorescence Staining of Transfected HeLa Cells
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HeLa cells were plated on coverslips and harvested at 48 h posttransfection. After being washed twice with phosphate-buffered saline (PBS), cells were fixed with 4% paraformaldehyde diluted by PBS and permeabilized using 0.1% Triton X-100. Then fixed cells were washed five times with PBS and incubated in blocking solution for 2 h. After washing off the blocking solution, cells were incubated with anti-HA (1:200; cat. no. H6908, Sigma) and anti-Flag (1:100; cat. no. F1804, Sigma) diluted in PBS containing 0.1% bovine serum albumin (BSA) for 2 h. Cells were washed three times with PBST and incubated with fluorescein (FITC) goat anti-mouse IgG (1:500; cat. no. 115-095-003, Jackson ImmunoResearch) and rhodamine (TRITC) goat anti-rabbit IgG (1:500; cat. no. 111-025-003, Jackson ImmunoResearch) for 1 h. Following four washes with PBS, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 1:2,000), and mounted on slides with Antifade Mounting Medium (Beyotime) for confocal microscopy analysis. The images were captured by Leica TCS SP5 confocal system.
3
Antibody Dilutions for WB and IF
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Antibodies (dilutions in parentheses) for Western blotting (WB) or indirect immunofluorescence (IF) were as follows: anti-Nopp140 rabbit serum (RS8 at 1:5000 for WB and 1:1000 for IF) (Kittur et al. 2007 (link)), mouse monoclonal anti-NAP57 immunoglobulin G (IgG) (H3 at 1:500 for IF; Santa Cruz Biotechnology), mouse monoclonal anti-β-actin (AC-15 at 1:1000 for WB; Santa Cruz Biotechnologies), mouse anti-γ-tubulin ascites fluid (GTU-88 at 1:5000 for WB; Sigma), DyLight488 goat antimouse IgG (1:500 for IF) and rhodamine (TRITC) goat antirabbit IgG (1:500 for IF; both Jackson ImmunoResearch), Alexa Fluor 680 goat antirabbit IgG (1:10,000 for WB; Thermo Fisher Scientific), and IRDyeTM 800 goat anti-mouse IgG (1:10,000 for WB; Rockland Immunochemicals).
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