Alexa fluor 568 conjugated donkey anti goat igg

Dissociated cochlear cells were fixed with 4% paraformaldehyde for 30 min. After three washes with 0.01 M PBS, the cells were incubated in blocking solution (10% goat serum in PBS with 0.25% Triton X-100) for 30 min. The cells were then incubated with anti-GLUT or anti-prestin antibodies (1 : 100–250) in blocking solution at room temperature (23°C) for 2 h. In control experiments, the primary antibody was omitted. After three washes with PBS, the cells were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1 : 200; cat. no. A21206, Molecular Probes) or Alexa Fluor 568-conjugated donkey anti-goat IgG (1 : 200; cat. no. A11057, Molecular Probes) in blocking solution at room temperature for 1 h. For costaining with di-8-ANEPPS to visualize the PM and cytoplasmic membranous organelles, the cells were further incubated in 30 μM di-8-ANEPPS (D-3167; Molecular Probes) for 20 min after secondary antibody incubation. After completely washing out the dye with 0.01 M PBS, staining was observed under a confocal microscope.
Cell nuclei were stained with Hoechst 33342 (R37605; Molecular Probes). Following incubation with the secondary antibody, pieces of dissociated cells were incubated with a dilution of Hoechst 33342 stock solution (2 drops per ml) at room temperature for 15–30 min and washed three times with PBS.

Frozen colon sections were treated with 0.5% Triton X-100 in Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS⁻) for 1 h and with blocking reagents containing 3% bovine serum albumin (BSA) for 1 h. Afterwards, they were incubated with goat polyclonal anti-MCP-1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with Alexa Fluor 568-conjugated donkey anti-goat IgG (Molecular Probes Invitrogen, Carlsbad, CA, USA) or phycoerythrin-conjugated monoclonal anti-CCR2 antibody (R&D Systems, Minneapolis, MN, USA). Nuclei were stained with TO-PRO3 iodide (Life Technologies, Eugene, OR, USA). Staining without anti-MCP-1 antibody, followed by incubation with Alexa Fluor 568-conjugated donkey anti-goat IgG or with phycoerythrin-conjugated rat IgG2b (BioLegend, San Diego, CA, USA), was performed as a negative control. The sections were examined using an Olympus IX81 FV1000 laser scanning confocal microscope.

Forty-eight hours after transfection, CHO cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 20 min and permeabilized with 0.2% Triton X-100 in PBS (PBST) for 10–20 min at room temperature. Cells were blocked with 10% Blocking One (Nacalai, Japan) in PBST for 30 min and then incubated overnight at 4 °C with rabbit polyclonal anti-Kv7.1 antibody (1:5000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and goat polyclonal anti-Kv11.1 antibody (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Following incubation, cells were labeled with an AlexaFluor^® 488-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, Oregon) at 1:400 dilution for KCNQ1 or with an AlexaFluor^® 568-conjugated donkey anti-goat IgG (Molecular Probes, Eugene, Oregon) at 1:400 dilution for hERG. Nuclei were stained with 4′-6-diamino-2-phenylindole (DAPI). Immunofluorescence stained cells were captured using a confocal laser scanning system Clsi (Nikon) on an Eclipse TE2000-E inverted microscope (Nikon). In the present study, if a cell showed KCNQ1 expression, above 90% of such cells simultaneously showed hERG expression in co-expression experiments.

Biopsy samples from the transverse colon were taken from macroscopically normal areas, transported to the laboratory in IVOC medium and processed within the next hour. IVOC was performed as described previously [74 ]. Briefly, biopsies were mounted on foam supports in 12 well plates and incubated with 30 μl EHEC standing overnight culture (approximately 10⁷ bacteria) and 200 nM of TD4 or Nb control (Vamy). Samples were incubated for 8 h on a rocking platform at 37°C in a 5% CO₂ atmosphere with medium changes after 4 and 6 h of incubation. At the end of the experiment, tissues were washed in PBS to remove the mucus layer and fixed in 3.7% formaldehyde/PBS for 20 min at RT. Samples were permeabilised with 0.1% Triton X-100/PBS, and blocked with 0.5% BSA/PBS for 20 min. Tissues were incubated with goat polyclonal anti-E. coli (1:400, Abcam) for one hour, followed by incubation in Alexa Fluor 568-conjugated donkey anti-goat IgG (1:400,ThermoFisher Scientific) and DAPI for 30 min to counterstain cell nuclei. Biopsy samples were mounted with Vectashield mounting medium (Vector Labs) and analysed using an Axio Imager M2 motorized fluorescence microscope (Zeiss). EHEC colonisation of colonic biopsies was quantified by counting adherent bacteria in a surface area of 1 mm².