Cells were fixed with 4% paraformaldehyde /4% sucrose for 30 min at room temperature and incubated for 1 h in blocking buffer (PBS containing 5% BSA, 5% normal goat serum, and 0.1% Triton X-100). The cells were then incubated with primary antibodies in blocking buffer at 4°C overnight. Primary antibodies included mouse monoclonal antibody against tyrosine hydroxylase (TH; 1:500; Sigma), rabbit polyclonal antibody against TH (1:50,000; Pel-Freez, Rogers, AR), rabbit polyclonal antibody against phospho-JNK (1:100; Cell Signaling), and rabbit polyclonal antibody against cleaved caspase 3 (1:1000; Cell Signaling). After three washes with PBS, the cells were incubated at room temperature for one hour with appropriate secondary antibodies: Alexa Fluor 488 (or 568) goat anti-rabbit IgG and Alexa Fluor 568 (or 488) goat anti-mouse IgG (1:200; Molecular Probes, Eugene, OR). Stained cells were monitored under a fluorescence microscope (Leica, Heidelberg, Germany).
Alexa fluor 488 or 568 goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 (or 568) goat anti-rabbit IgG is a secondary antibody conjugated with a fluorescent dye. It is designed to bind to rabbit primary antibodies, allowing for the detection and visualization of target proteins or antigens in various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 568 or 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Rabbit polyclonal antibody against cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Prolong antifade, by Thermo Fisher Scientific (1 mentions)
Radiance 2000, by Zeiss (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Cathepsin b, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole dilactate dapi, by Thermo Fisher Scientific (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
Dm il led, by Leica (1 mentions)
5 protocols using alexa fluor 488 or 568 goat anti rabbit igg
1
Immunofluorescence Analysis of Neuronal Markers
2
Brain Immunohistochemistry of Dopaminergic Neurons
3
Immunostaining of Dopaminergic Neurons
4
Detecting Autophagic Vesicle Colocalization
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Huh7 cells infected at greater than 90% were transfected with different plasmids as indicated in figure legends. At 24 h post-transfection, cells were trypsinized and grown on glass coverslips for another 24 h. The coverslips were then fixed with 4% formaldehyde in PBS for 10 min, washed in PBS and incubated in blocking buffer (PBS, 3% bovine serum albumin, 10% FBS, 0.1% Triton X-100) for 30 min at RT. For the detection of GFP-LC3, cells were permeabilized with 0.05% saponin to remove dispersed LC3 (LC3-I) [27 (link)]. After washing with PBS, the coverslips were incubated with primary antibodies in blocking buffer for 1 h at RT. Coverslips were then washed in PBS and incubated with either Alexa fluor-(488 or 568) goat anti-mouse IgG or Alexa fluor-(488 or 568) goat anti-rabbit IgG (Invitrogen) for 1h at RT. After washing, coverslips were mounted on glass slides with Prolong Antifade (Invitrogen) and examined with either a laser scanning confocal BioRad Radiance 2000 or a Zeiss LSM 780. The Manders’coefficient of colocalization was obtained using ImageJ software (NIH) in randomly selected regions that were positive for the targeted proteins from different cells. Manders’coefficient values over 0.4 were considered as strong colocalization.
5
Immunofluorescence Imaging of Mammary Tissue
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Mammary glands were fixed, embedded in paraffin and sectioned (5 μm) as described previously54 (link). Tissues sections were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich; St. Louis, MO, USA) in PBS (pH 7.4) for 45 min or ice-cold 100% methanol for 10 min and blocked in 10% goat serum, 0.05% Tween-20 (Sigma-Aldrich). Primary antibodies were pStat3 (1:400; Cell Signaling; Beverly, MA, USA) and cathepsin B (1:50; Santa Cruz; Dallas, TX, USA) and secondary antibodies were Alexa Fluor 488 or 568 goat anti-rabbit IgG (1:1,000; Invitrogen). HA and Lamp1 were detected with mouse anti-HA IgG, Alexa Fluor 488 conjugate (1:1,000; Invitrogen) and Alexa Fluor 647 anti-mouse Lamp1 (1 μg/mL; BioLegend, San Diego, CA, USA). Nuclei were stained with 4′, 6-Diamidino-2-Phenylindole, dilactate (DAPI; 1:20; Invitrogen). For cultured cells, coverslips were fixed in 4% w/v phosphate buffered-paraformaldehyde (pH 7.4) for 10 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 10 min. The coverslips were then blocked with 5% goat serum in PBS for 20 min. Slides were mounted in ProLong Gold (Invitrogen) and imaged on an Olympus FV1000 (Olympus; Center Valley, PA, USA) as described below or by light microscopy (Leica DM IL LED, Leica Microsystems GmbH; Wetzlar, Germany) and images were captured using Leica Application Suite (version 3.6.0).
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