Flow cytometry was performed as described previously (Ishikawa et al., 2012 (link)) with a slight modification. In brief, bacterial cells were resuspended in PBS containing 4% (w/v) paraformaldehyde and incubated at room temperature for 15 min. The samples were washed with PBS and treated with anti-AtaA699-1014 (Ishikawa et al., 2012 (link)) or anti-AtaA59-325 (Aoki et al., 2020 (link)) rabbit antiserum at a 1:10000 dilution in PBS containing 0.05% (v/v) Tween 20. After a 30-min incubation at room temperature, the samples were washed twice with NET buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, 0.05% (v/v) Triton X-100, pH 7.6) and treated with Alexa Fluor 488-conjugated anti-rabbit antibody (Cell Signaling Technology, MA) at a 1:500 dilution in NET buffer for 30 min. The samples were washed twice with NET buffer and resuspended in deionized water, and the fluorescence was measured by FACS Canto II (Becton, Dickinson and Company, NJ, United States). Histograms were created using Flowjo software (Tomy Digital Biology, Tokyo, Japan).
Alexa fluor 488 conjugated anti rabbit antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Alexa Fluor 488-conjugated anti-rabbit antibody is a secondary antibody used for detection and visualization in various immunological and cell biology applications. It is designed to bind to primary rabbit antibodies, allowing for the specific labeling and detection of target proteins or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
Histoclear, by National Diagnostics (1 mentions)
Anti mouse antibodies, by Cell Signaling Technology (1 mentions)
Fluorsave reagent, by Merck Group (1 mentions)
Bl ac40ts c2, by Olympus (1 mentions)
5 protocols using alexa fluor 488 conjugated anti rabbit antibody
1
Flow Cytometry Analysis of AtaA Antigens
2
Quantifying Bacterial Cell Immobilization
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Bacterial cells before and after the cell immobilization/detachment test were resuspended in PBS containing 4% paraformaldehyde and incubated at room temperature for 15 min. The samples were washed with PBS and treated with anti-AtaA699–1014 antiserum diluted 1:10,000 in PBS. After a 1-h incubation at room temperature, the samples were washed twice with NET buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris–HCl, 0.05% Triton X-100, pH 7.6) and treated with Alexa Fluor 488-conjugated anti-rabbit antibody (Cell Signaling Technology, MA) diluted 1:500 in NET buffer for 30 min. Finally, the samples were resuspended in dH2O, and the fluorescence was measured by FACS Canto II (Becton, Dickinson and Company, NJ).
3
Quantification of Enterobacteriaceae and DNA Damage in Colon Tissue
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Paraffin-embedded, transverse sections of colon tissue were hybridized overnight with 2 µM fluorescence in situ hybridization probe specific for Enterobacteriaceae members at 55°C in hybridization buffer (Kempf et al., 2000 (link)). The slides were subsequently washed three times with fluorescence in situ hybridization wash buffer and blocked with 4% bovine serum albumin in PBS for 30 min at RT. Blocked slides were stained with anti-phosphorylated γ-H2AX antibody (#9718S; Cell Signaling) and subsequently with Alexa Fluor 488–conjugated anti-rabbit antibody (#4412; Cell Signaling). Tile images were captured using an LSM780 confocal microscope (Zeiss) and stitched using Imaris software 8.2 (Bitplane AG). Quantification of tissue-associated E. coli burden as well DNA damage was performed using the spot and surface function of Imaris software 8.2. In each tissue, 6,688 to 23,809 cells were counted, and only fluorescent objects with a diameter >6 µm (average diameter of mammalian cell nucleus) were counted to eliminate contamination and nonspecific staining.
4
Cyclodextrin-mediated Stilbene Delivery
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Hydroxypropyl-β-cyclodextrin was kindly provided by Roquette Frères (Lestrem, France). RES (3,5,4′-trihydroxystilbene) was purchased from APIChem Technology (Hangzhou, China). NNK was purchased from Toronto Research Chemicals (Toronto, Canada). Mouse anti-γ-H2AX (Ser139) antibody and FluorSave reagent were purchased from Merck Millipore (Billerica, MA, USA). Rabbit anti-p65 antibody, Alexa Fluor® 488 conjugated anti-rabbit antibody, HRP-linked anti-rabbit and anti-mouse antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Fluorescein isothiocyanate (FITC) conjugated anti-mouse antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Normal goat serum was purchased from Abcam (Cambridge, UK). Histo-clear was purchased from National Diagnostics (Atlanta, GA, USA). Other chemicals were purchased from Sigma-Aldrich (Saint-Louis, MO, USA), and used without further purification.
5
Force Mapping and Flow Cytometry Analysis
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Force mapping was performed using NanoWizard 3 BioScience AFM system (JPK Ins., Berlin, Germany) with Advanced QI mode, which is an extensional software of QI [27] mode at RT in PBS, PBS supplemented with 1% casamino acid technical (Becton, Dickinson and company, Franklin Lakes, NJ, USA), PBS supplemented with 1% glucose, 100 mM KCl solution, or DW. Clean silicon-probe AFM cantilevers with spring constants of 0.02-0.14 N/m (BL-AC40TS-C2; Olympus Ltd., Tokyo, Japan) or gold-coated probe cantilevers with spring constants of 0.003-0.13 N/m (HQ:CSC38/Cr-Au-B; MikroMasch, Madrid, Spain) were used. The spring constants of the cantilevers were determined using the thermal noise method. The parameters used in QI mode are the following: Z-length: 3 m; applied force: 0.2 nN; speed: 20 m/s.
Flow cytometry was performed as described previously [30] . AtaA on the cell surface was immune-stained with anti-AtaA699-1014 antiserum and Alexa Fluor 488-conjugated antirabbit antibody (Cell Signaling Technology, Danvers, MA, USA) and detected using a flow cytometry system (FACS Canto II; Becton, Dickinson and company).
Flow cytometry was performed as described previously [30] . AtaA on the cell surface was immune-stained with anti-AtaA699-1014 antiserum and Alexa Fluor 488-conjugated antirabbit antibody (Cell Signaling Technology, Danvers, MA, USA) and detected using a flow cytometry system (FACS Canto II; Becton, Dickinson and company).
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