For whole-mount embryo analysis, embryos were fixed with 4% paraformaldehyde (PFA). β-Galactosidase activity in the Rosa26R3 crosses was detected after incubation with X-gal (5-bromo-4-chloro-3-indolyl-b-d -galactopyranoside; Invitrogen). Alkaline phosphatase activity in Z/AP mice was detected using BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; Vector Laboratories) staining according to manufacturer’s protocol. Cre expression in the uterus was evaluated using the Gt (ROSA)26R3Sortm1Sor/J, Z/AP, and mT/mG mouse reporter lines. Frozen sections were fixed with 4% PFA and then stained with BCIP/NBT or by immunofluorescent analysis for mT or mG. Immunofluorescence analysis was performed on paraffin-embedded samples as previously described (17 (link)).
The effects of Lifr deficiency on glial cells of the central nervous system (CNS) and osteoclasts in bone were histologically characterized and by hematoxylin and eosin (H&E) staining with images being recorded on a Zeiss Axioimager. Paraffin-embedded thin sections of spinal cords (5 μm) were stained with an antibody against GFAP to identify glial cells and counterstained with Nissl stain. The osteoclasts in the long bones were identified by their unique morphology and size after H&E staining, performed using standard histological procedures.
The effects of Lifr deficiency on glial cells of the central nervous system (CNS) and osteoclasts in bone were histologically characterized and by hematoxylin and eosin (H&E) staining with images being recorded on a Zeiss Axioimager. Paraffin-embedded thin sections of spinal cords (5 μm) were stained with an antibody against GFAP to identify glial cells and counterstained with Nissl stain. The osteoclasts in the long bones were identified by their unique morphology and size after H&E staining, performed using standard histological procedures.