Astrocytes were cultured on coverslips in 24-well plates, and the cells were stimulated with 100 ng/mL LPS. After LPS stimulation, 50% of the cultures were treated with 200 ng/mL Atsttrin for 24 hours (Wei et al., 2017). Immunofluorescence was performed following a published protocol with minor modification (Tarassishin et al., 2014). Primary antibodies (rabbit anti-cyclooxygenase-2 (COX-2) and rabbit anti-i-NOS, 1:100, Abcam) were added to the cells and incubated overnight at 4°C. The cells were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:300, Cell Signaling Technology, Danvers, MA, USA) at 37°C for 1 hour, followed by incubation with 4′,6-diamidino-2-phenylindole for 5 minutes. An IX-81 fluorescence microscope was used to capture images.
Alexa fluor 488 conjugated goat anti rabbit secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody is a fluorescently labeled antibody that binds to rabbit primary antibodies. It is designed for use in immunoassays and other applications where detection of rabbit antibodies is required.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti inos, by Abcam (1 mentions)
Mps 60, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Alexa fluor 488 conjugated goat anti mouse secondary antibody, by Absin (1 mentions)
Dapi staining, by BioLegend (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
4 protocols using alexa fluor 488 conjugated goat anti rabbit secondary antibody
1
Atsttrin Modulates Astrocyte Inflammation
2
Immunofluorescent Staining of Ki-67 and PIKfyve
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Immunofluorescent (IF) staining was performed as previously described.24 (link) Cell plates were washed by PBS for 3 times, fixed with 4% paraformaldehyde for 30 minutes, and then blocked in 1% blocking solution. Plates were then incubated in dark with primary antibody against Ki-67 (cat 9129S, 1: 200; Cell Signaling Technology, Danvers, MA) at 4°C overnight. VSMC plates were then washed and subsequently stained with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (cat 4412S, 1: 2500; Cell Signaling Technology) in dark for 1 hour. Next, VSMC plates were stained with DAPI (cat MX4208, 5 mg/mL; VECTOR Labs, Burlingame, CA) for 5 minutes in room temperature. For PIKfyve staining, the artery sections were incubated with anti-PIKfyve antibody (cat sc-100408, 1: 50) and then followed by a secondary antibody (1:300; Invitrogen). Nuclei were stained with DAPI. Images were obtained by using an IF microscopy (Leica MPS 60; Wetzlar, HD, Germany). The fluorescence intensity was measured by using Image J software (Bethesda, MA).
3
Multiparametric Tumor Microenvironment Analysis
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The frozen tumors were cut into 10μm sections for immunofluorescence staining and confocal imaging. Staining for blood vessels was achieved by incubation with anti-CD31 mouse mAb conjugated to Alexa Fluor®488 (dilution 1:200, Santa Cruz, USA). Anti-smooth muscle actin mouse mAb conjugated to Alexa Fluor®647 (dilution 1:200, Santa Cruz, USA) was used for detection of α-SMA. Collagen I was detected with the anti-COL1A1 mouse mAb (dilution 1:200, Santa Cruz, USA) and Alexa Fluor®488-conjugated goat anti-mouse secondary antibody (dilution 1:500, Absin, China). HIF-1α was detected with the anti-HIF-1α rabbit mAb (dilution 1:200, Cell Signaling Technology, USA) and Alexa Fluor®488-conjugated goat anti-rabbit secondary antibody (dilution 1:500, Cell Signaling Technology, USA). PD1 was detected with the anti-PD1 rabbit mAb (dilution 1:200, Cell Signaling Technology, USA) and Alexa Fluor®647-conjugated goat anti-rabbit secondary antibody (dilution 1:500, Cell Signaling Technology, USA). Cell nuclei were marked with DAPI staining (dilution 1:5000, Biolegend, USA). The images were captured at 20× magnification via confocal microscopic examination. The intensity values of the aim indicators were quantified with Image J software.
4
Visualization of EGFR Expression in Lung and Colon Cancer Cells
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NCI-H460 and SW620 cells (1 × 105 cells) were cultured on the top of a cover-slip slide for 24 h at 37°C. The medium was replaced with fresh serum free medium (500 μL) containing Tc-99m SYPIPDT-ECG-TAMRA (200 nM). Cells were then incubated at 37°C for 1 h. These cells were rinsed three times with phosphate-buffered saline (PBS). The cell were stained with anti-human wtEGFR antibody (cat. 4267S; Cell Signaling Technology, Danvers, MA, USA) and Alexa Fluor® 488-conjugated goat anti-rabbit secondary antibody (cat. 111-545-003, 1:100 dilution; Jackson Immuno Research Inc., West Grove, PA, USA). The slides were covered with fluorescent mounting medium (Dako, Glostrup, Denmark) and closed with cover glasses. The confocal microscopic study was performed with a FV1200 confocal microscope (Olympus, Pittsburgh, PA, USA) with a 100× oil immersion lens.
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