Nitroblue tetrazolium nbt

Salmon louse were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 24 hours and transferred to 70% ethanol at 4°C for at least 24 hours before paraffin embedding. In situ hybridization was carried out according to [41 (link)], with the same modifications as described in [42 (link)]. Digoxigenin (DIG)-labelled anti-sense and sense RNA probes (Lsnvd: 261 bp, Lsshd: 327 bp, Lsdib: 475 bp) were generated from PCR templates (Table 1) with T7 promoters using DIG RNA labelling kit (Roche Diagnostics GMbH, Mannheim Germany). 25 ng/μl probe was used in the hybridisation mix for detection of Lsshd, Lsdib and Lsnvd, respectively. Chromogenesis was carried out using nitroblue tetrazolium (NBT) (Roche Diagnostics GMbH) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche Diagnostics GMbH). Sense RNA was used as a negative control.

To obtain fetal tissues, timed breeding of heterozygous/homozygous SCL‐3′Enh‐PLAP transgenic mice was established. Fetuses were obtained from day 10 (E10) to day 14 (E14) of gestation. Cell preparation procedures from embryonic/fetal and adult hematopoietic tissues were previously published 35, 43, (Supporting Information). A fragment of yolk sac (YS) was used for embryo typing by staining with the PLAP substrate, nitroblue tetrazolium (NBT) (Roche, Mannheim, Germany, www.lifescience.roche.com) as described 39. LSEC enriched nonparenchymal cell (NPC) fraction was obtained from adult mouse livers by a two‐step collagenase perfusion technique and gradient percoll centrifugation as described 26, 29 with modifications (Supporting Information).