Rnascope technology

In situ RNA hybridization for Fndc4 mRNA was performed on formaldehyde-fixed, paraffin-embedded colonic tissue using RNAscope technology (Advanced Cell Diagnostics, Hayward, CA, USA) with custom-made probes for mouse Fndc4, following the manufacturer‘s instructions (RNAscope 2.0 HD red detection kit). Probes targeting Ubc and Dabp were used as positive and negative control probes, respectively. Slides were counterstained with hematoxylin.

In situ RNA hybridization was performed using RNAscope technology (Advanced Cell Diagnostics, Hayward, CA, USA) following the manufacturer’s protocol, as described (Pronin et al., 2014 (link)). Briefly, at 12 h postinjury, experimental and control eyes were dissected, formalin fixed and embedded in optimal cutting temperature (OCT) medium. Whole-fixed mouse eyes were cut into 10 μm sections and mounted on SuperFrost Plus glass slides. After removing the OCT medium with PBS, slides were treated for 15 min with boiling pretreatment 2 solution, followed by pretreatment 3 (protease) for 30 min at 37°C. To reduce chromosomal DNA background, we introduced a DNase treatment step: slides were washed 5× with water and treated with DNase I (50 U/ml in 1× DNase I buffer, Ambion) for 40 min at 37°C. To demonstrate that the signal comes from hybridization of probes with mRNA, some slides were treated with a mixture of DNase I and RNase A (5 mg/ml). Following 5× wash with water, slides were hybridized with RNAscope probes for 2 h at 40°C. Following in situ RNA hybridization steps, regular immunohistochemistry was performed to colocalize transcript signals with retinal cell markers: RBPMS for RGCs, GFAP for astrocytes, glutamine synthetase for Muller glia and Iba1 for microglial cells. The fluorescent signals were visualized and captured using a Leica SP5 confocal microscope.

Hearts from AB- or sham-operated mice were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. 7 μm paraffin sections were cut and Adamtsl3 mRNA was detected using in-situ hybridization with the RNAscope technology (Advanced Cell Diagnostics, Cat. No. 465521). A HybEZ oven was used for hybridization and the 2.5 HD Red detection kit was used for visualization. The tissue was counterstained with hematoxylin. An Olympus BX51 microscope (Olympus, Center Valley, PA) with a Leica DFC7000T camera was used for imaging with the Leica Application Suite v4.6 software.

RNAScope^® technology (Advanced Cell Diagnostics Milan, Italy) was used for in situ RNA hybridization according to the manufacturer's protocol. The sections were washed in 1x PBS + 0.1% triton for 3 × 5 min and then placed on glass slides and air dried. The slides were incubated in warmed pre-treatment 3 (Advanced Cell Diagnostics Milan, Italy) for 5 min, kept at a light boil, and then placed in distilled water. The slides were then subjected to RNAscope^® Multiplex Fluorescent Assay by first incubating the probe (mmOlig2, mmCasp3, or mm MOG) for 2 h at 40°C and then pre-forming the amplification steps according to instructions. The slides used for Olig2/EdU labeling were then blocked with 5% normal goat serum in 1x PBS +0.1% triton at room temperature for 1 h, and the Click-iT^® assay (described above) was then performed followed staining with the nuclear marker DAPI for 5 min. The sections were washed and mounted (Everbrite Hardset mounting medium, Biotium, Hayward, CA).