TUBO cells were grown to 80% confluence on sterile round glass coverslips in a six well tissue culture plate. Cells were washed three times with PBS and fixed in 4% (wt/vol) paraformaldehyde (Fisher Scientific, Cat. No. 50-980-487) for 15 min. Next, cells were permeabilized with 0.2% Triton X (Sigma Aldrich, Cat. No. T8787) for 10 min and washed three times with PBS. After washing, cells attached to cover slips were incubated with 5% (wt/vol) bovine serum albumin (BSA) (Fisher Scientific, Cat. No. BP1605) in PBS for 1 h at room temperature. Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark. After being washed with PBS three times, coverslips were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Cat. No. H-1200). The stained coverslips were examined and imaged using a Zeiss Apotome.2 fluorescence microscope (Carl Zeiss Inc., Thornwood, NY).
Alexa fluor 594 conjugated anti rabbit secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Alexa Fluor 594-conjugated anti-rabbit secondary antibody is a fluorescently labeled detection reagent used to visualize and locate rabbit primary antibodies in various applications, such as immunofluorescence and Western blotting. The Alexa Fluor 594 dye provides a bright red fluorescent signal that can be detected using appropriate filter sets.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Triton x, by Merck Group (1 mentions)
Apotome 2 fluorescence microscope, by Zeiss (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Smz1500 microscope, by Nikon (1 mentions)
Inverted a1r spectral confocal microscope, by Nikon (1 mentions)
3 protocols using alexa fluor 594 conjugated anti rabbit secondary antibody
1
Immunofluorescence Staining of HER2 in TUBO Cells
2
Immunofluorescence Staining of Stem Cell Markers
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The cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich)/Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific) for 20 min at room temperature, permeabilizedwith 0.2% Triton X-100/PBS for 5 min and then subjected to blocking in 10% FBS/DPBS for 20 min at room temperature. For CD44 and CD34 staining, the cells were incubated with anti-CD44 (Clone DB105, #130-113-334, 1:100 dilution) and anti-CD34 (Clone AC136, #130-113-178, 1:100 dilution) FITC antibodies (Miltenyi Biotec Inc., Auburn, CA, USA) overnight at 4°C, and washed 3 times in PBS. For hypoxia-inducible factor (HIF)-1α (cat. no. 3716) and VEGFc (cat. no. 2445) (both from Cell Signaling Technology, Danvers, MA, USA) the cells were first stained with primary antibody (1:100 dilution, overnight at 4°C) followed by washes 3 times with PBS and again incubated with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (cat. no. A-11012; Thermo Fisher Scientific; 1:200 dilution, overnight at 4°C).
3
Confocal Imaging of Dorsal Aorta
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Images were obtained with a Nikon SMZ1500 microscope or a Nikon inverted A1r spectral confocal microscope. Confocal z-stack image acquisition in multiple fluorescent channels was used to examine the entire dorsal aorta in the AGM . Immunofluorescence double staining was performed as described previously,58 (link) with anti-p-akt S473 (1:25; cat. no. 9271; Cell Signaling) and AlexaFluor 594–conjugated anti-rabbit secondary antibody (1:1000; cat. no. A11012; Life Technologies).
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