Alkaline phosphatase conjugated sheep anti digoxigenin fab fragment

Spinal cords from human embryos at 46 days of gestation were fixed in 4% PFA (par-formaldehyde), paraffin embedded and sectioned (7 μm thickness). PAD2 and PAD3 probes were produced by PCR amplification of the regions corresponding to exon 1 and exon 7 of PAD2 and PAD3 cDNAs, and ligation into the pGEMT-Easy vector (Promega). The pGEMT-Easy vectors containing either the PAD2 or PAD3 insert were digested with SpeI or NcoI restriction endonuclease and transcribed using T7 and Sp6 RNA polymerase to produce DIG labeled riboprobes.
In situ hybridization was carried out using digoxigenin-labeled riboprobes essentially as previously described [38] (link). In brief, de-waxed sections digested with proteinase K (20 mg/ml) were re-fixed in 4% PFA solution, treated with 0.1 M triethanolamine containing 0.25% acetic anhydride and hybridized overnight at 65°°C. After high stringency washes, the riboprobes were localized using an alkaline phosphatase-conjugated sheep anti-digoxigenin Fab fragment (Roche) and detected by incubation in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Roche).

Embryos were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight, rinsed with PBST (0.1% Triton X-100 in PBS), transferred to methanol, and stored at −20°C until use. After rehydration with PBST, embryos were incubated with proteinase K solution. Embryos were refixed with 4% PFA for 20 min and then washed with PBST four times. Embryos were then pre-hybridised and hybridised at 65–67°C with antisense RNA probes. Subsequently, non-binding antisense RNA probes were washed, and embryos were transferred to blocking solution (5% sheep serum) for 2 hr at room temperature (RT). An alkaline phosphatase-conjugated sheep anti-digoxigenin Fab′ fragment (Roche, Basel, Switzerland) was added (1:4000 dilution) and incubated for 3 hr. Embryos were then washed five times with PBST for 15 min each at RT and then 2 times for 15 min each with staining buffer. Next, BCIP/NBT was added, and embryos were incubated at RT until staining.