Cells were cultured on coverslips and transfected with specific expression plasmids, then fixed and permeabilized with 4% paraformaldehyde (Sigma-Aldrich) for 15 min, and then washed three times with PBS followed by 0.1% Triton X-100 (Sigma-Aldrich) for 10 min. After washing, the cells were blocked using 5% bovine serum albumin for 1 h at 37°C and then incubated with primary antibody (anti-Myc) diluted in PBS containing 5% BSA for 1 h at 37°C. The cells were then washed three times and incubated with a fluorescently labeled secondary antibody (Alexa-Fluor 568-conjugated donkey anti-mouse IgG antibody; Thermo Fisher Scientific; catalog no.A10037) for 1 h at 37°C in the dark. After the cells were washed three times with PBS, they were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology; catalog no. C1002) at room temperature for 5 min. The cells were observed under a confocal immunofluorescence microscope (Carl Zeiss, Oberkochen, Germany).
Confocal immunofluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Confocal immunofluorescence microscope is an instrument used for high-resolution imaging of fluorescently labeled samples. It operates by scanning the sample with a focused laser beam, and collecting the emitted fluorescence light through a pinhole aperture, which enhances the image contrast and resolution.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Alexa fluor 568 conjugated donkey anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
3 protocols using confocal immunofluorescence microscope
1
Immunofluorescence Microscopy of Transfected Cells
2
Co-Immunoprecipitation and Confocal Imaging of Protein Interactions
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293T or HeLa cells were transfected with the indicated plasmids. Co-IP and confocal microscopy were performed as described previously [25 (link)]. Briefly, for IP assay, 293T cell lysates were collected and centrifuged. The supernatants were then incubated with antibody-bound Dynabeads Protein G (Life Technologies, 10004D) for 20 min at room temperature. The Dynabeads were then washed with ice-cold PBS containing 0.1% Tween-20 (MACKLIN, T885238) and eluted in 1× SDS-PAGE sample buffer. Then proteins were analyzed by western blotting with the indicated antibodies. For confocal microscopy, HeLa cells were first fixed with PBS supplemented with 4% paraformaldehyde (Sigma-Aldrich, P6148) at room temperature for 15 min. The cells were then washed and permeabilized with PBS containing 0.1% Triton X-100 (Sigma-Aldrich, T9284) at 4 °C for 20 min. Next, cells were blocked with PBS supplemented with 5% bovine serum albumin (BSA; Yeasen, 36101ES25). After that, the cells were incubated with the primary antibodies (anti-Flag tag or anti-HA tag) at 37 °C for 1 h, washed five times with PBS, and incubated with the secondary antibodies at 37 °C for 1 h. After washing five times, the cells were examined using a confocal immunofluorescence microscope (Carl Zeiss, Oberkochen, Germany).
3
Immunofluorescence Staining of Paraffin Sections
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After deparaffinisation, hydration and washing, the paraffin sections were subjected to high‐pressure antigen retrieval using citrate buffer (pH 6.0). The Triton buffer (.25% Triton X‐100 in phosphate‐buffered saline) used to permeate sections. After blocking the sections with goat serum, the primary antibodies were incubated overnight at 4°C, and the secondary antibodies were incubated for 1 h at room temperature the following day. Sections were mounted with the medium containing 4',6‐diamidino‐2‐phenylindole (DAPI) for fluorescence (Cat# ZLI‐9557, ZSGB‐BIO). A same operation was performed on cells. Images were scanned with a confocal immunofluorescence microscope (Carl Zeiss). The primary antibodies used in the experiment are shown in Table S2 .
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