In Situ Hybridization of Adamts4 and Syt1

In situ hybridization (ISH) was performed on 16 µm coronal cryosectioned tissues as previously described. The antisense riboprobe generation of full length of adamts4 and syt1 were described previously (Su et al., 2010 (link); Levy et al., 2015 (link)). Briefly, riboprobes were synthesized using digoxigenin (DIG)-labeled UTP (Roche, Mannheim, Germany) and the MAXIscript In Vitro Transcription Kit (Ambion, Austin, TX, USA). Probes were hydrolyzed to 500 nt. Sagittal brain sections were prepared and hybridized at 65℃ as previously described (Su et al., 2010) (link), and bound riboprobes were detected by horseradish peroxidase (POD)-conjugated anti-DIG antibodies and fluorescent staining with Tyramide Signal Amplification (TSA) systems (PerkinElmer, Shelton, CT, USA). Images were obtained on a Zeiss Axio Imager A2 fluorescent microscope or a Zeiss Examiner Z1 LSM 700 confocal microscope. A minimum of three animals per genotype and age was compared in ISH experiments.