Anti digoxigenin fab fragments antibody

A 614 bp digoxin-conjugated complementary RNA (cRNA) probe (targeting Gfap mRNA) was prepared using SP6 RNA polymerase-mediated in vitro transcription. The DNA template for in vitro transcription was prepared using Gfap mRNA-specific forward primers 5′-GTGGATTTGGAGAGAAAGGTTG-3′ and reverse primer 5′- GCGATTTAGGTGACACTATAGCTGGAGGTTGGAGAAAGTCTGT-3′ (underlined is the core sequence recognized by SP6 RNA polymerase). The in vitro transcription was performed and used as templates according to the instructions of DIG RNA labeling Kit (SP6/T7, Cat# 11175025910, Roche). Frozen sections (14 μm thickness) were used for mRNA ISH. The hybridization was performed at 65°C for 12 to 18 h. The sections were washed by washing solution containing saline sodium citrate buffer (SSC) three times 10 min per time and immersed in RNases solution at 37°C for 30 min to eliminate non-specific binding and non-hybridized mRNAs. The sections were then incubated in NBT/BCIP solution (Cat# 11681451001, Roche) according to the manufacturer’s instruction at 4°C overnight followed by blocking with blocking buffer for 1 h at room temperature and incubated with anti-Digoxigenin Fab fragments antibody (Cat#11093274910, Roche) at 4°C overnight. The DIG-conjugated mRNA signals were visualized by HNPP/Fast Red Fluorescent Detection set (Cat# 11758888001, Roche) according to the kit instructions.