Hnpp fast red fluorescent detection set

In situ hybridization was performed as previously described [11] (link). Briefly, animals were sacrificed by cervical dislocation and fresh brain tissue was dissected out, embedded in Tissue Tek (Sakura), frozen on dry ice and stored at −80°C until used. 14 µm cryostat sections were cut and hybridization was performed according to protocols using either digoxigenin-, fluorescein- or ³⁵S-labelled antisense RNA probes. In digoxigenin-labelled insitu hybridization experiments, NBT/BCIP (Roche) was used as substrate for alkaline phosphatase, while in radioactive insitu hybridization sections were exposed to Biomax MR X-ray films (Kodak) for two to seven days. For double ISH, sections were hybridized simultaneously with DIG- and fluorescein-labelled probes. A two-step chromogenic reaction using NBT/BCIP and HNPP/Fast Red Fluorescent Detection Set (Roche) was performed to visualize DIG- and fluorescein-labelled riboprobes. Specimens were counterstained with DAPI.

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.

Whole-mount ISH (WISH) was performed according to Kawaue et al. [19 (link)] by the use of a semiautomatic ISH machine (HS-5100; Aloka, Tokyo, Japan) (Figure 1A–D’, E). The ISH on sections was performed according to Sato et al. [20 (link)]. Each RNA probe was used at a concentration of 0.67 μg/mL (WISH) or 0.17 μg/mL (sections) in hybridization buffer. Fluorescence ISH using HNPP/Fast Red Fluorescent Detection set (Roche #11758888001) was performed basically according to the kit instructions but without proteinase treatment for subsequent immunofluorescence (Figure 2B–D). ISH experiments were performed at least in triplicate (Figure 1) or in duplicate (other data).