Colon cancer cell lines SW620 were plated as monolayers in black clear bottom 96 well plates at 20,000 cells/well in 100 μL of phenol red free medium. After cells attached, various concentrations of neo, 7 and etoposide were added for 6 or 24 h. Cells were fixed with 4% paraformaldehyde at room temp for 20 min, washed 3 times with 400 μL/well of PBS, permeabilized and blocked with 100 μL of 3% BSA and 0.1% IGEPAL in PBS for 20 min. Cells were then incubated in γ-H2AX primary antibody (Cell Signaling, MA, 1:400 dilution) at 4 °C overnight, washed 4 times with PBS, and incubated with FITC conjugated anti-rabbit secondary antibody (Cell Signaling, MA, 1:200 dilution) at room temp for 1 h, and washed 4 times with PBS. Cells were stained with 10 μM of Hoechst 33342 at room temp for 10 min, washed 4 times with PBS before imaged with a 20x objective using PerkinElmer Operetta imager.
Fitc conjugated anti rabbit secondary antibody
Manufactured by Cell Signaling Technology
FITC conjugated anti-rabbit secondary antibody is a laboratory reagent used to detect and visualize rabbit primary antibodies in various immunoassays. It consists of a fluorescein isothiocyanate (FITC) molecule conjugated to an antibody that binds to rabbit immunoglobulins.
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2 protocols using fitc conjugated anti rabbit secondary antibody
1
Evaluating DNA Damage in Colon Cancer Cells
2
HER2 Immunofluorescence Staining Protocol
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For HER2 IF staining, sections were washed twice with buffer (1× TBS/0.1% Tween-20). Endogenous peroxides were blocked with BLOXALL (SP-6000, Vector Labs, Burlingame, CA). Tumor tissues were washed 2× with buffer solution, then incubated with blocking solution (5% Normal Goat Serum/wash buffer) for 1 h at room temperature and washed again. HER2 (#2165S, Cell Signaling, Danvers, MA) primary antibody (1:400 dilution) in blocking solution was added, and sections incubated overnight at 4 °C in a humidified chamber, followed by multiple washes and addition of FITC conjugated anti-rabbit secondary antibody (Cell Signaling, #4412) at a dilution of 1:300 in blocking solution. Tissue sections were incubated at room temperature for 30 min and washed in triplicate for 5 min. Nuclear counterstaining was performed with DAPI (0.1 mg mL−1). Sections were washed twice for 5 min, followed by coverslip application using Prolong Gold Antifade (ThermoFisher, #P10144) mounting media.
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