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12 protocols using cy3 anti mouse secondary antibody

1

Immunofluorescence Staining of Key Signaling Proteins

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For PI (3,4,5)P3, PTEN, USP11, or FOXO1 immunofluorescence staining, cells were incubated with anti-PI(3,4,5)P3 antibody, anti-PTEN, anti-USP11, or anti-FOXO1. Cells were then washed and incubated with Cy3 anti-mouse secondary antibody (1:5000; Jackson ImmunoResearch) for 1 h. Slides were then mounted in Vectashield containing 4′,6-diamidino-2-phenylindole (Vector Lab). Immunofluorescence images were generated using a Leica DMI 3000B fluorescence microscope (Leica).
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2

Immunofluorescence Assay for PML Protein

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Cells (5 × 106/well in 12-well plates) were seeded in 1 mL RPMI-1640 supplemented with 5% FBS overnight and washed with 1x PBS twice on next day. Cells were cultured with 0.5 mL serum-free RPMI for another 24 h Ovcar3 cells were then treated 1 μM KU60019 and 10 μM fenofibrate; Ovcar10 cells were treated 10 μM KU60019 and 25 μM fenofibrate in RPMI + 0.1% FBS for 3 days. KU60019 was administered 5 h prior to fenofibrate. Cells were fixed in 4% paraformaldehyde for 10min and permeabilized in 0.2% Triton X-100 for 5 min. Cells then were stained with PML (1:200, Santa Cruz, Cat# sc-966) in 3% BSA/PBS at room temperature for 1 h. Cells were further incubated with 0.15 μg/ml DAPI in PBS (1 min), mounted, and sealed. Cells were washed three times and then incubated in Cy3 anti-mouse secondary antibody (1:5000, Jackson ImmunoResearch Labs, Cat# 715-165-150) in 3% BSA/PBS at room temperature for 1 h. Finally, cells were incubated with 0.15 μg/ml DAPI in PBS for 1 min, washed three times with PBS, mounted, and sealed. Images were acquired at room temperature using a Nikon Eclipse 90i microscope with a 20x/0.17 objective (Nikon DIC N2 Plan Apo) equipped with a CoolSNAP Photometrics camera.
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3

Immunofluorescence Analysis of αSMA Expression

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Hs68, PCF54 and PCF55 cells were seeded in DMEM-F12 complete medium at a density of 1 × 104 cells per well in 24-well plates on glass coverslips and grown as a monolayer for 24 h. After that, the cells were rinsed with PBS, fixed, and permeabilized with methanol-acetone (1:1) in -20 °C for 20 min, then washed with PBS and blocked with 2% BSA. Cells were incubated with αSMA primary antibody (sc-32,251, Santa Cruz Biotechnology, USA; 1:50 dilution in PBS, 1% BSA) overnight in + 4 °C and with Cy3-anti mouse secondary antibody (115-165-071, Jackson Immunoresearch, UK) for 1 h in room temperature in dark. Next, cells were washed with PBS and mounted on glass slides in ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, USA) and incubated in + 4˚C, overnight. Fluorescence imaging was done on Leica DM3000 microscope (Leica Microsystems GmbH, Germany).
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4

Immunofluorescent Staining of Vascular Cells

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Before immunostaining, cells were permeabilized for 2 min at room temperature in 0.5% Triton X-100 (Sigma-Aldrich) in 4% paraformaldehyde solution. Cells were then washed three times with PBS, incubated in 4% paraformaldehyde solution for 20 min at room temperature, and then probed with fluorophore-tagged antibodies. Samples were incubated with antibodies targeting VE-cadherin (Cayman Chemical, rabbit; 1:100) and smooth muscle α-actin (Santa Cruz Biotechnology, mouse; 1:25) prepared in blocking buffer (0.1% bovine serum albumin in universal buffer) for 1 hour in 37°C. Samples were then incubated in secondary antibody (Alexa Fluor 488 anti-rabbit secondary antibody or Cy3 anti-mouse secondary antibody, Jackson ImmunoResearch; 1:100 dilution) for 1 hour in 37°C. A Zeiss LSM 780 or a Zeiss LSM 800 laser scanning confocal microscope was used for confocal imaging. Cell viability was measured using calcein-AM and ethidium homodimer-1 staining and imaged with a Zeiss LSM 780.
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5

Vinculin Immunofluorescence Staining

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Cells were then washed with warm 1×
phosphate-buffered saline (PBS) twice before being fixed with 4% formaldehyde
at room temperature for 15 min followed by permeabilization with a
solution of 0.1% Triton X-100 in PBS at room temperature for 5 min.
To block nonspecific binding, the samples were incubated in 1% bovine
serum albumin (BSA) in PBS for 1 h at room temperature. After blocking,
an anti-vinculin mouse primary antibody at a dilution of 1:500 in
1% BSA in PBS was added to the samples and incubated for 1 h at room
temperature. The samples were consequently washed with 0.5% Tween
20 in PBS three times for 5 min. A Cy3 anti-mouse secondary antibody
(1:200) (Jackson ImmunoResearch Laboratories Inc.), Phalloidin Alexa
Fluor 488 (1:100) (Thermofisher Scientific, U.K.) in 1% BSA in PBS,
was consequently added to the samples and incubated for 1 h at room
temperature followed by another three 5 min washes with 0.5% Tween
20 in PBS. The nuclei of the cells were stained using Vectorshield-DAPI
(Vector Laboratories) before being imaged with a fluorescent microscope
(Zeiss, GmbH, Germany). All reagents unless otherwise stated were
sourced from Sigma-Aldrich, U.K.
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6

Immunostaining of Embryonic Samples

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One-day-old adults were incubated in 1x M9 solution for 4–5 hours and their embryos were removed, fixed, and stained77 (link). Embryos were attached to microscope slides coated with poly-lysine and containing Teflon spacers. Slides were frozen on dry ice, embryos were permeabilized by freeze-crack method and submerged in 100% MeOH for 5–10 minutes at - 20°C. Embryos were submerged for 5 minutes twice in PBS, then once in PBT (PBS plus 0.1% Tween). Embryos were incubated in primary antibody overnight at 4°C. Embryos were then washed in PBT for 5 minutes three times and then incubated in secondary antibody for 1 hour at 37°C. Embryos were washed once in PBT then twice in PBS, mounted in Vectashield (Vector Laboratories), and stored at 4°C. The following primary antibodies were used: anti-HA primary antibody (Abcam, 1:200), anti-Myc (Abcam, 1:100), anti-GFP (Abcam, 1:200), anti-PAR-3 (DSHB), anti-GIP-1 (GenScript7 (link)). The following secondary antibodies were used: CY3-anti-mouse secondary antibody (Jackson Immunoresearch Laboratories, 1:200), Streptavidin Alexa Fluor 488 (Invitrogen, 1:200), DAPI (Sigma, 1:10,000), 647-anti-rabbit (Jackson Immunoresearch Laboratories, 1:50), 488-anti-mouse (Jackson Immunoresearch Laboratories, 1:200), 488 anti-rabbit (Jackson Immunoresearch Laboratories, 1:200), Cy5 anti-mouse (Jackson Immunoresearch Laboratories, 1:50).
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7

Immunostaining of Drosophila Brain

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Flies were collected and brains dissected in phosphate-buffered saline (PBS), and then fixed in 4% PFA at RT for 30 mins, blocked with 5% normal goat serum (NGS) in PBST (0.3% Trition X-100 in PBS) at RT for 1 hr. Samples were incubated with primary at 4C overnight, and washed 3 3 15 min in PBST. Secondary antibody incubation (Jackson ImmunoResearch Inc.) was performed at R.T. for 1 hour, then washed 3 3 15 min with PBST and mounted with Vectashield Mounting Medium (H-1000, Vector Laboratories). Imaging was performed using a Leica TCS SP8 microscope. Anti-Fasciclin II was used at 1:20 (1D4, Developmental Studies Hybridoma Bank). Anti-mouse Cy3 secondary antibody was used at 1:500 (Cat# 515-165-003, Jackson ImmunoResearch).
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8

Immunostaining of Drosophila Brain

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Flies were collected and brains dissected in phosphate-buffered saline (PBS), and then fixed in 4% PFA at RT for 30 mins, blocked with 5% normal goat serum (NGS) in PBST (0.3% Trition X-100 in PBS) at RT for 1 hr. Samples were incubated with primary at 4C overnight, and washed 3 3 15 min in PBST. Secondary antibody incubation (Jackson ImmunoResearch Inc.) was performed at R.T. for 1 hour, then washed 3 3 15 min with PBST and mounted with Vectashield Mounting Medium (H-1000, Vector Laboratories). Imaging was performed using a Leica TCS SP8 microscope. Anti-Fasciclin II was used at 1:20 (1D4, Developmental Studies Hybridoma Bank). Anti-mouse Cy3 secondary antibody was used at 1:500 (Cat# 515-165-003, Jackson ImmunoResearch).
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9

Quantifying Muscle Stem Cell Proliferation

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Six days prior to harvesting, mice were given the thymidine analogue 5-Bromo-2′-deoxyuridine (BrdU, 1 mg/ml, Sigma, B5002) in the drinking water supplemented with sucrose (25 mg/ml). Muscle stem cells were isolated by FACS, plated on µ-Slide 8 Well (Ibidi, 80826) in medium (40% DMEM, 40% MCDB (Sigma), 20% FBS, 2% Ultroser (Pall), 1% Penicillin-Streptomycin (ThermoFisher)) at 37 °C 5% CO2 3% O2 to allow adherence, fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) for 5 min at RT, washed twice 5 min with PBS, permeabilised in 0.5% Triton X-100 (Sigma) for 5 min at RT, washed three times with PBS, incubated with DNAse I (Roche, 04536282001) at 1 unit/µL in PBS at 37 °C for 30 min, washed three times with PBS, blocked in 10% goat serum (GS, Gibco) for 30 min at RT, incubated with mouse anti-BrdU antibody (1/100, BD, 347580) in 2% GS for 2 h at RT, washed three times 5 min in PBS, incubated with anti-mouse Cy3 secondary antibody (1/500, Jackson ImmunoResearch, 115-165-205) and Hoechst (1 µg/ml) in 2% GS for 45 min at RT, and washed four times with PBS. Images were taken with a Zeiss LSM800 confocal (×40 objective).
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10

Immunostaining of Cytochrome c in Neurons

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The status of cyt c (whether intact in the mitochondria or released) was examined by immunostaining with cyt c antibodies. Briefly, sympathetic neurons were cultured for 4 days after which they were either left untreated or deprived of NGF for various time points. Neurons and other cell lines were fixed in 4% paraformaldehyde and incubated overnight in anti-cyt c primary antibody (556432, BD Biosciences) followed by a 2 h incubation with anti-mouse Cy3 secondary antibody (Jackson Labs). Nuclei were stained with Hoechst 33258 (Molecular Probes).
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