miR-142-5p Disrupts Neuronal Morphogenesis Underlying Porcine Hemagglutinating Encephalomyelitis Virus Infection by Targeting Ulk1

Li, Zi; Lan, Yungang; Zhao, Kui; Lv, Xiaoling; Ding, Ning; Lu, Huijun; Zhang, Jing; Yue, Huiqing; Shi, Junchao; Song, Deguang; Gao, Feng; He, Wenqi

In situ hybridization of endogenous mRNAs and microRNAs was performed as described (Wibrand et al., 2010) . Briefly, tissue sections and primary cortical neurons were fixed with 4% paraformaldehyde/DEPC-PBS. After pre-hybridization in hybridization buffer at 55 • C for 2 h, hybridization with digoxigenin (DIG)-labeled mRNA probes or biotin-labeled microRNA fluorescence in situ hybridization (FISH) probes (EXONBIO, Guangzhou, China) was performed at 42 • C overnight. Subsequently, blocking reagent was applied, followed by incubation with an aminomethylcoumarin (AMCA)conjugated anti-DIG or rhodamine-conjugated anti-biotin antibody (EXONBIO, Guangzhou, China) at 37 • C for 30 min together with a MAP2 rabbit mAb (in the dark). After counterstaining the samples with 4' ,6-Diamidino-2-phenylindole (DAPI)-Antifade at room temperature for 20 min, the slides were examined under a fluorescence microscope with a proper filter set.