Rnaiso plus 9109

Total RNAs in callus tissues of the intervertebral bone graft fusion site of mice were extracted using RNAiso plus 9109 following the manufacturer’s protocol (Takara, Tokyo, Japan). Complementary DNA (cDNA) was synthesized using 2 μg of total RNA as template, with a Hifair II 1st Strand cDNA synthesis kit (Cat. No. 11121ES60) as described by the manufacturer’s protocol (Yeasen Biotech., Shanghai, China). Target gene transcriptions, including α smooth muscle actin (α-SMA), collagen-1 (Col-1), osteocalcin (OCN), osteopontin (OPN), and bone sialoprotein (BSP), were amplified using a Hieff UNICON Universal Blue Qpcr SYBR Green Master Mix (Cat. No. 11184ES03, Yeasen Biotech., Shanghai, China) and determined using an AB Step One plus Real-Time PCR System (Applied Biosystems, Foster city, CA, USA). The reaction conditions of the RT-PCR assay were listed as follows: 95 °C for 2 min, and 40 cycles involving 95 °C for 10 s and 60 °C for 30 s. Transcriptions of the above targeting genes were calculated according to the description of a previously published 2^−ΔΔCt method, by normalizing to the housekeeping gene GAPDH (Livak and Schmittgen, 2001 (link)). The RT-PCR primer sequences are shown in Table.

Fluorescein‐conjugated dextran (D3306, molecular weight: 3 kDa, Dex‐3) was purchased from Invitrogen. Gd‐DPTA (molecular weight: 938 Da) was obtained from CONSUN. HiLyte Fluor‐488‐labeled recombinant human α‐syn (AS‐55457, molecular weight: 14 kDa HiLyte‐488‐α‐syn) was purchased from Anaspec. Adeno‐associated virus expressing A53T‐α‐syn (AAV‐A53T) was designed by OBio Technology. Primary antibody anti‐AQP4 (rabbit, AB3594), anti‐α‐syn (mouse, 36–008; Syn211), and anti‐tyrosine hydroxylase (rabbit, AB152; anti‐TH) were purchased from Millipore. Anti‐PDGF‐B (rabbit, DF6328) was purchased from Affinity Biosciences, and anti‐PDGFRb (mouse, sc‐374573) was purchased from Santa Cruz Biotechnology. β‐actin (mouse, ab008‐100), horseradish peroxidase‐conjugated anti‐rabbit and anti‐mouse IgG (70‐gam007, 70‐gar007) were purchased from Multisciences. Secondary antibodies, including Cy3‐conjugated donkey anti‐rabbit and Cy5‐conjugated donkey anti‐rabbit, were purchased from Jackson ImmunoResearch. DNaseI and 4′,6‐diamidine‐2′‐phenylindole dihydrochloride (DAPI) were purchased from Sigma‐Aldrich. RNAiso Plus (9109), PrimeScrip RT Master Mix (RR036A) and SYBR Premix Ex Taq II (RR820A) were purchased from TaKaRa.