HeLa cells were plated on glass slides a day after transfection and immunostained the next day as follows: fixed in 4% PFA for 15 min, permeabilized in 0.5% Triton X-100 in PBS for 10 min, blocked in 0.1% Triton X-100 in PBS with goat serum (1:80) for 30 min, stained with primary antibody in 0.1% Triton X-100 in PBS with goat serum (1:100) for 1 hour at 37 degrees C, washed three times in 0.1% Triton X-100 in PBS for 5 min, stained with secondary antibody in 0.1% Triton X-100 in PBS for 1 hour, washed three times in 0.1% Triton X-100 in PBS for 5 min, mounted in Vectashield mounting media (VECTOR) with DAPI. Antibodies used: SRSF1 (1:500, Abcam, ab38017), FLAG (1:500, Sigma, F1804), Alexa Flour 488 goat anti-rabbit IgG and Alexa Flour 594 goat anti-mouse IgG (1:500, Thermo Fisher Scientific). Leica TCS SP5-II confocal microscope on inverted stand, with 63x/1.4 NA oil objective, was used to take single optical slices in DAPI, Alexa488 and Alexa594 channels.
Alexa flour 594 goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594 goat anti-mouse IgG is a secondary antibody conjugated with the Alexa Fluor 594 fluorescent dye. It is designed to detect and visualize mouse immunoglobulin G (IgG) in various immunoassays.
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4 protocols using alexa flour 594 goat anti mouse igg
1
Immunocytochemistry for SRSF1 and FLAG
2
Immunocytochemistry for SRSF1 and FLAG
3
Endocytic Trafficking of Elastin-Like Polypeptides
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MDA MB231 cells were seeded on a cover glass inside a 6-well chambered dish filled with appropriate culture media. After 24 h, cells were incubated with Alexa-488-labeled respective ELPs (0.3125 μM) for 1 h at 37 °C. Cells were washed with PBS and further incubated with pre-warmed culture medium containing lysotracker deep red (Molecular Probes, Life Technologies Corporation, Eugene, OR, USA). Later, the cells were fixed with 4 % paraformaldehyde, nuclei-stained with Hoechst, and observed under a confocal microscope. For early endosome staining, cells were seeded on a 4-well chambered slide to 80% confluence. Cells were then treated with respective ELPs for 30 min or 1 h. Early endosomes were stained by anti-EEA1 (BD Biosciences, Franklin Lakes, NJ, USA) for 1 h at 37 °C, followed by Alexa Flour-594 goat anti-mouse IgG (Life Technologies Corporation, Eugene, OR, USA) secondary antibody for 1 h. The cells were observed under a confocal microscope after nuclei were stained with Hoechst.
4
Quantifying siRNA Uptake into Early Endosomes
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MDA-MB231 cells (8 × 104) were seeded in 4-chamber slide and incubated with anti-Early endosome antigen 1 (BD Biosciences, Franklyn Lakes, NJ, USA) diluted in DMEM (Dulbecco’s Modified Eagle Medium) for 1 h at 37 °C. Cells were then washed with PBS several times to remove unbound antibody and then incubated with Alexa Flour-594 goat anti-mouse IgG (Life Technologies Corporation, Eugene, OR, USA) secondary antibody and siRNA/ELP variant complexes (50 pmol NC-siRNA labeled with Alexa 488) for 1 h. Cells were then fixed with 4% paraformaldehyde and nuclei were stained with Hoechst 33342. The internalizations of siRNA/ELP complexes into early endosomes were assessed by confocal microscopy (LSM700; Carl Zeiss).
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