Alexa fluor 488 conjugated streptavidin

Biotin-labeled RNA probes targeting circIPO7 back-splice junctions were designed and synthesized by GenePharma (Shanghai, China). GC cells were grown to 50–75% confluence at the time of fixation with 4% paraformaldehyde and hybridized with circIPO7 probes at 37 °C overnight in hybridization buffer (10% formamide, 10% dextran sulfate, 2×SSC), followed by incubation with Alexa Fluor 488-conjugated streptavidin (Invitrogen, USA) in the dark for 1 h. Finally, the cells were counterstained with DAPI (Invitrogen, USA) for nuclear visualization. Signals were detected under a Zeiss LSM880 confocal microscope (Leica Microsystems, Germany). The sequences of the probes were as follows: circIPO7, 5’-GGUGUGAUAUAUUCUUUAGCUCUUUAUGAUCUUCAAC-3’; random control 5’-AGCUGAAUCAUGAGAACCAGAUC-3’.

Immunofluorescence staining was performed using previously described methods.^{52 (link)} Briefly, frozen sections of the spleen were fixed with cold acetone for 10 min at 4 °C and blocked with 5% normal goat serum for 30 min. Sections were then stained with biotin-conjugated PNA (RL-1072; Vector), Alexa Fluor 647-conjugated IgD (11–26c.2a; eBioscience) and PE-conjugated CD4 (RM4–5; BD Biosciences) antibodies, followed by Alexa Fluor 488-conjugated streptavidin (25–4317–82; Invitrogen). Cover slips on which both types of cells were cultured were mounted on slides with the ProLong Antifade Kit (P-7481; Life Technologies) and examined under a Zeiss LSM 510 confocal fluorescence microscope. The images were processed with ImageJ software.

To visualize the CST, the anterograde tracer biotinylated dextran amine (BDA; 10 000 MW; dilution, 10% in PBS; Invitrogen) was slowly injected with a glass capillary attached to a micro-syringe into the forelimb motor area as determined by a functional map of the motor cortex (coordinates from bregma: 0 mm anterior/1.0 mm lateral, 0.5 mm anterior/1.0 mm lateral, 0 mm anterior/1.5 mm lateral, and 0.5 mm anterior/1.5 mm lateral, 0.4 μl per site, all at a depth of 0.5 mm into cortex).^{37 (link)}Two weeks after BDA injection, we obtained transverse cryosections of spinal cord from C4 to C6. For visualization of BDA-labeled fibers, the sections were treated with 0.3% Triton-X-100 in PBS for 1 h, followed by incubation with Alexa Fluor 488-conjugated streptavidin (1 : 400; Invitrogen) in PBS for 2 h. To quantify the number of crossing axons, we counted the number of BDA-positive fibers crossing from the intact side of the spinal cord to the contralateral side in 10–20 sections for each mouse. To normalize for differences in the tracing efficiency of the individual animals, the number of fibers crossing the midline was divided by the total number of labeled main CST fibers in the dorsal column.

Anterograde-tracing was performed to detect axonal regeneration as previously described [21 (link)]. Biotinylated dextran amine (BDA; MW,10000; Invitrogen) 10% w/v in sterile PBS was injected into two sites (0.4 μl/ site) of the spinal cord by micropipette. One week later, mice were anesthetized and perfused transcardially with 4% paraformaldehyde. Thirty-micron sections were incubated with Alexafluor 488-conjugated streptavidin (1:500; Invitrogen) for 1 h at room temperature. The images were acquired with a confocal microscope (Olympus, BX51).

To examine the biodistribution of E. coli in mice, tumors and other normal organs were removed and imaged on an IVIS Spectrum 8 days after i.v. administration of 4 x 10⁷ c.f.u. E. coli (lux) or E. coli (lux/βG). Samples were homogenized and spread on carbenicillin-containing LB plates to count bacterial colonies. Spleen weights from each group were measured. For immunofluorescence histology, tumor samples were fixed with 4% paraformaldehyde in PBS and embedded with paraffin. Sections were stained with goat anti-E. coli polyclonal antibody (Abcam, Cambridge, MA) and biotinylated 1E8 (anti-E. coli beta-glucuronidase) followed by rhodamine-conjugated rabbit anti-goat IgG (Organon Teknika Corporation, West Chester, PA) and Alexa Fluor 488 conjugated streptavidin (Invitrogen), respectively. Sections were incubated with 1 μg/ml DAPI to visualize nuclei.

Cells grown on tissue culture dishes were washed with PBS and harvested with Accutase cell dissociation solution (Sigma-Aldrich) for 5 min at 37 °C. Cells (1 × 10⁶ cells/mL) were then re-suspended in ice-cold PBS pH 7.4/1% FBS (staining buffer) and blocked in ice-cold PBS pH 7.4/5% FBS for 10 min at 4 °C, followed by two washes in staining buffer. Cells were stained with AlexaFluor 488-conjugated Streptavidin (Invitrogen) or primary antibody for 1 h at 4 °C and washed twice with staining buffer. Primary antibodies targeted against TGFβR1/ALK-5 (1:500) is from Novus Biologicals; Integrin β1-Biotin conjugated (Ha2/5) (1:250) from BD Biosciences. Additional staining was done using AlexaFluor 488-conjugated secondary antibody for 30 min at 4 °C. For the quantification of apoptosis, adherent cells along with those in culture media were collected and washed twice with cold PBS. Cells were stained with PE-Annexin V (BD Biosciences), according to the manufacturers’ instructions, and analyzed immediately by flow cytometry. Samples were acquired using BD FACS Canto II instrument and BD FACS Diva v8.0.2 software. Data were analyzed using FlowJo v10 software, and represented as geometric mean of fluorescence is represented as Mean Fluorescence Intensity (MFI) or percentage (%) of Annexin V positive cells.