Alexa 488 conjugated donkey anti rabbit igg secondary antibody

ssON’s interaction with nucleolin expressed on the surface of cells was assessed using a modified version of the flow cytometry-based cell surface binding assay (9 (link)). A549 cells were detached with 2mM EDTA, chilled on ice and pre-incubated with ssON (1 µM) for 30 min at 4°C, followed by incubation with anti-nucleolin antibody (Ab22758, Abcam) at different concentrations for 1h at 4°C and then washed. The binding of the anti-nucleolin antibody to the cell surface, was revealed by staining with Alexa 488-conjugated donkey anti-rabbit IgG secondary antibody (5 µg/ml; Invitrogen) for 1h at RT. Cells were then washed and the data acquisitions were obtained using a FACSVerse (BD) and all analyses were performed with FlowJo software (Tree Star).

The nucleolin binding assay was performed as previously described with minor modifications [14 (link),24 (link)]. Briefly, 2 mM EDTA was used to detach the cells and they were put on ice to cool. All of the treatments occurred at 4 °C to ensure that no cellular uptake could occur. The cells were treated with RNA or DNA oligonucleotides (1 µM) for 15 min prior to incubation with an anti-nucleolin antibody (Ab22758, Abcam, Cambridge, UK) for 30 min at 4 °C, at a concentration of 1 µg. Subsequently, the cells were stained with a secondary Alexa 488-conjugated donkey anti-rabbit IgG secondary antibody (5 µg/mL; Invitrogen, Massachusetts, MA, USA) for 30 min at 4 °C, followed by the acquisition of the samples using a FACSVerse cytometer (BD, New Jersey, NJ, USA). The analysis was performed using FlowJo software (Tree Star). Alternatively, after the secondary antibody staining, the cells were placed in imaging dishes and analyzed on a Zeiss Axio Observer Z1 microscope using a 20× objective followed by analysis using Zen Blue 3.2.

For Dbn1 mRNA detection in fibrotic hearts and livers, we used RNAscope Multiplex Fluorescent Reagent kit v2 (ACD) as per the manufacturer’s instructions. The fibrotic heart and liver were fixed with 4% PFA overnight, incubated in 20% sucrose (Nacalai Tesque), and embedded in optimal cutting temperature compound (Sakura Finetek). Next, the heart and liver samples were sectioned into 6 and 10 μm thick sections, respectively, and used for RNAscope analysis
For performing Tnni3 or Desmin costaining after staining for Dbn1 mRNA, the sections were blocked with 10% BSA in PBS for 1 h and incubated at 4 °C overnight with goat anti-Tnni3 antibody (1:200; Abcam; ab56357) or rabbit anti-Desmin antibody (1:200; Abcam; ab32362) diluted with 10% BSA in PBS. Then, the cells were incubated at 20 °C for 1 h with Alexa488-conjugated donkey anti-goat IgG secondary antibody (1:200; Abcam; ab150129) or Alexa488-conjugated donkey anti-rabbit IgG secondary antibody (1:200; Invitrogen; A21206) diluted with 10% BSA in PBS. The cells were then washed with PBS and incubated with 4′,6-diamidino-2-phenylindole (1:1000; Dojindo) in PBS at 20 °C for 5 min, following which they were mounted with FluorSave reagent and observed using a confocal microscope (LSM700).