Resource Summary Report  New Search Previous Search Results

URL: https://dx.doi.org/10.17504/protocols.io.j9hcr36

Authors: Michiyo Tsuru

Group: Clinical Proteomics and Gene Therapy Laboratory

Summary: We generated a miR-340 probe by tagging Has-miR-340 aatcaG(L)t5(L)aT(L)tG(L)cT(L)ttataa_N(6)_Y with Alexa Fluor 488. LNA-in situ hybridization (ISH) was performed according to the manufacturer’s instructions (http://www.exiqon.com/mirna-ish-kit). Sections of FFPE tissues of human OPLL and null mice (4-µm-thick) were fixed with 4 % PFA in PBS for 20 min at room temperature, washed with PBS (3 × 5 min), treated with 0.5 % Triton X-100 (10 min at 4 °С), and briefly washed with PBS followed by two washes (10 min each) in saline-sodium citrate buffer (2× SSC, 0.3 M NaCl, 0.03 M Na3С6Н5О7, рН 7.0). The sections were then digested with 15 μg/mL proteinase K (Exiqon, Vedbaek, Denmark) at 37 °C for 10 min and rinsed for 3 × 5 min in PBS. Hybridization was performed in a humid chamber for 18 h at 65 °C. In situ hybridization of miR-340 was conducted using a miRCURY LNA microRNA ISH Optimization kit (Exiqon, Vedbaek, Denmark). miR-340-Alexa Fluor 488 and bone morphogenetic protein 2 (Alexa Fluor 555) were detected in human OPLL tissue by in situ hybridization and immunocytochemistry, respectively. Nuclei were counterstained with DAPI. After staining, the tissues were observed by fluorescence microscopy (BZ-X700, Keyence, Osaka, Japan).

Associated Publications: Tsuru M, Ono A, Umeyama H, Takeuchi M, Nagata K (2018) Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification. PLoS ONE 13(5): e0196204. doi: 10.1371/journal.pone.0196204

Affiliations: Clinical Proteomics and Gene Therapy Laboratory, Kurume University, Fukuoka, Japan

External URL: https://doi.org/10.1371/journal.pone.0196204

Version: 1

Publication Date: 2018

Expand All Collapse