Autosomal Recessive Hypercholesterolemia Caused by Mutations in a Putative LDL Receptor Adaptor Protein

, 5374 (1999).24. Cells were Þxed in 4% paraformaldehyde/0.15% picricacid in phosphate-buffered saline (PBS). Immunocyto-chemistry was carried out with the use of standardprotocols. The following primary antibodies were usedat following dilutions: nestin rabbit polyclonal 1:500(made in our laboratory), TUJ1 mouse monoclonal1:500 and TUJ1 rabbit polyclonal 1:2000 (both fromBabco, Richmond, CA), insulin mouse monoclonal1:1000 (Sigma, St. Louis, MO), insulin guinea pig poly-clonal 1:100 (DAKO, Carpinteria, CA), glucagon rabbitpolyclonal 1:75 (DAKO), somatostatin rabbit polyclonal1:100 (DiaSorin, Stillwater, MN), GFP 1:750 polyclonal(Molecular Probes, Eugene, OR), and BRDU rat mono-clonal 1:100 (Accurate, antibodies, Westbury, NY). Fordetection of primary antibodies, suorescently labeledsecondary antibodies (Jackson Immunoresearch Labo-ratories, West Grove, PA, and Molecular Probes) wereused according to methods recommended by the man-ufacturers. Histochemical staining for alkaline phospha-tase cells was carried out using commercially availablekit (Sigma) following manufacturerOs recommendations.25. A. Ferreira, A. Caceres,