Cells (2 × 105 cells per well) were cultured on the cell climbing films. The prepared cell climbing films were fixed by 4% PFA (Sinopharm, China) for 15 min, permeabilized with 0.1% TritonX-100 (Beyotime) for 30 min at room temperature, and pre-incubated in 1% BSA (Sangon, China) for 15 min at room temperature. For γH2AX staining, the cell climbing films were incubated overnight at 4°C with antibodies against γH2AX (1:50, Thermofisher, #PA5-97354) and then incubated for 60 min at room temperature with Alexa Fluor™ 555-labeled goat anti-rabbit IgG (1:200; Invitrogen, #A27039; Red). For SH3RF2-RBPMS staining, the cell climbing films were incubated overnight at 4°C with antibodies against SH3RF2 (1:100, Thermofisher, #PA5-63527) and RBPMS (1:50, Santa cruz, #sc-293285) and then incubated for 60 min at room temperature with FITC-labeled goat anti-rabbit IgG (1:200; Abcam, UK, #ab6717; Green) and Alexa Fluor™ 555-labeled goat anti-mouse IgG (1:200; Invitrogen, #A-21424; Red). The cell nuclei were counterstained with DAPI (Aladdin, China). The images were captured with a fluorescence microscope (Olympus, Japan).
Alexa fluor 555 labeled goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Alexa Fluor™ 555-labeled goat anti-rabbit IgG is a secondary antibody used for fluorescent detection of rabbit primary antibodies in various immunoassay applications. It is conjugated with the Alexa Fluor™ 555 dye, which emits in the orange-red region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 labeled goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 labeled goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Sinopharm (1 mentions)
Nb100 92156, by Novus Biologicals (1 mentions)
Slowfade gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
Anti ha antibodies m20013, by Abmart (1 mentions)
Anti α tubulin ab15246, by Abcam (1 mentions)
Anti gfp antibodies g1544, by Abcam (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Inverted fluorescence microscope, by Zeiss (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
5 protocols using alexa fluor 555 labeled goat anti rabbit igg
1
Immunofluorescence Staining of Cellular Markers
2
Visualizing CFTR Expression in Mouse Intestine
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Briefly, the duodenum, defined as the first 3 cm of the small intestine, was dissected from wild type, untreated CF and CF mice treated by whole body cooling. The duodenum was fixed with 4 % paraformaldehyde solution and then embedded in paraffin before being sectioned. Paraffin-embedded mouse small intestine was sectioned in 5 μm thick slices. For anti-CFTR staining, sections were pretreated by heat-induced epitope retrieval, blocked in 10 % goat serum for 1 h and then incubated overnight with primary antibodies for CFTR (NB100-92156, Novus Biologicals) at 1:100 in incubation medium (PBS with 3 % goat serum, 3 % BSA, and 0.3 % Triton × 100). After four 5 min washes in washing buffer, the sections were incubated with secondary antibodies (Alexa Fluor 555–labeled goat anti-rabbit IgG; Invitrogen) for 1 h at room temperature at a concentration of 2 μg/ml in incubation medium. After four washes for five min each, cover slips were mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen). Some slides were co-stained with wheat germ agglutinin, Alexa Fluor® 488 conjugate (Invitrogen), to visualize mucin secreted by goblet cells in mouse intestine.
3
Zebrafish Embryo Cell Culture and Viral Propagation
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Zebrafish wild-type AB line was purchased from China Zebrafish Resource Center. Fish were raised with 10 h darkness and 14 h light at 28°C and were fed with commercial pellets twice a day. All embryos were obtained by natural spawning and staged as previously reported (16 (link)).
ZBE3 cells derived from zebrafish embryos were cultured at 28°C as previously described (17 (link)). HEK 293T cells were cultured in DMEM (Invitrogen) enriched with 10% FBS (Invitrogen) at 37°C under a humidified atmosphere of air containing 5% CO2.
RGNNV was propagated in ZBE3 cells and stored at −80°C until use.
Anti-Flag (M20008), anti-Myc (M20002), anti-His (M20001L), and anti-HA antibodies (M20013) were purchased from Abmart. Anti-α-tubulin (ab15246) and anti-GFP antibodies (G1544) were purchased from Abcam and Sigma, respectively. Goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, Alexa Fluor 488-labeled goat anti-mouse IgG, and Alexa Fluor 555-labeled goat anti-rabbit IgG secondary antibodies were purchased from Invitrogen.
ZBE3 cells derived from zebrafish embryos were cultured at 28°C as previously described (17 (link)). HEK 293T cells were cultured in DMEM (Invitrogen) enriched with 10% FBS (Invitrogen) at 37°C under a humidified atmosphere of air containing 5% CO2.
RGNNV was propagated in ZBE3 cells and stored at −80°C until use.
Anti-Flag (M20008), anti-Myc (M20002), anti-His (M20001L), and anti-HA antibodies (M20013) were purchased from Abmart. Anti-α-tubulin (ab15246) and anti-GFP antibodies (G1544) were purchased from Abcam and Sigma, respectively. Goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, Alexa Fluor 488-labeled goat anti-mouse IgG, and Alexa Fluor 555-labeled goat anti-rabbit IgG secondary antibodies were purchased from Invitrogen.
4
RGNNV Infection Visualization in GS Cells
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GS cells were grown on coverslips (10 mm × 10 mm) in six-well plates. The cells were pretreated with various reagents, such as the translation inhibitor cycloheximide (CHX), the PKR inhibitor 2-aminopurine (2-AP), or the PERK inhibitor GSK2606414) and then the cells were infected with RGNNV. Treated or untreated GS cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 1 h at room temperature. After washing with PBS, the cells were permeabilized with 0.2% triton X-100 for 15 min, blocked with 2% bovine serum albumin, and incubated with rabbit anti-G3BP1, mouse anti-CP, mouse anti-TIA-1, or mouse anti-eIF3η serum (1:200) for 2 h at 37°C. The cells were incubated with secondary antibody (Alexa Fluor 555-labeled goat anti-rabbit IgG or Alexa Fluor 488-labeled goat anti-mouse IgG (1:200; Molecular Probe) for 1 h, then cells were stained with 4, 6-diamidino-2-phenylindole (DAPI) for 5 min. The samples were observed under an inverted fluorescence microscope (Zeiss, Oberkochen, Germany).
5
Intracellular Localization of TTP and Ago2
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HeLa cells were split to a density of 1 × 105/well in six-well plates 24 h before transfection. On the following day, each cell sample was incubated for 6 h in a 1-ml transfection mixture containing 2 μl of Lipofectamine 2000 reagent (Invitrogen) and siRNAs at a concentration of 50 nM according to the manufacturer’s protocols. Twenty-four hours later, cells were divided and applied to chamber slides (1 × 105/well) and further incubated for 24 h. Seventy-five nanomolar siRNA, 500 ng of total plasmids, and 2 μl of Lipofectamine 2000 were mixed in 1 ml Opti-MEM medium in the second transfection. Plasmids included 50 ng of TTP-WT-EGFP or TTP-AA-EGFP, 100 ng of Myc-Ago2 or Myc-Septin1, and 150 ng of HA-tagged-MKK6b(E) or 6b(A). PcDNA3.0 was added to make a total of 500 ng of plasmid in each experiment. Forty-eight hours after transfection, cells were fixed in 4% paraformaldehyde for 15 min and permeabilized and blocked with PBS–1% goat serum–0.1% Triton X-100 for 30 min. For indirect immunofluorescence, all the primary and secondary antibodies were diluted 1:500 with 1% (wt/vol) bovine serum albumin (BSA) in PBS. PBs were detected using rabbit polyclonal anti-Dcp1a antibody and Alexa Fluor 555-labeled goat anti-rabbit IgG (Molecular Probes). Fluorescence was captured with a laser-scanning confocal microscope (Leica TCS SP5, Germary).
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