Tissue tek optimal cutting temperature compound

Levels of human serum albumin in transplanted mice were determined as previously described [33 (link)].
Livers, spleens and pancreases were harvested and fixed with 10% formalin for sectioning and immunofluorescence staining. Tissues were processed through a sucrose gradient (10%, 20%, 30% and 50% in PBS) and frozen in Tissue-Tek Optimal Cutting Temperature compound (Ted Pella Inc., Redding, CA). Cryo-blocks were sectioned on a Cryostat Leica CM1850 UV (Leica Biosystems Nussloch GmbH, Nussloch, Germany) and stained with primary and secondary antibodies as previously described [30 (link)].
Flow cytometry was used to analyse the engraftment of livers and spleens by human cells as previously described [30 (link),33 (link)]. Livers and spleens were digested with Life Technologies' Collagenase IV (Thermo Fisher Scientific). The light-density fractions of spleen cells were collected by centrifugation over a layer of Axis-Shield's Lymphoprep (STEMCELL Technologies Inc., Vancouver, BC). Flow cytometry was also used to analyse engraftment of human haematopoiesis in the central bone marrow, which was collected as previously described [35 (link)]. Staining with fluorescent antibodies was performed as described [28 (link),30 (link)].

Fetal liver sections were prepared, stained, and analyzed by epifluorescence microscopy as previously described (9 (link)). Briefly, livers were fixed with 10% formalin, processed through a sucrose gradient (10%, 20%, 30% and 50% in PBS) and frozen in Tissue-Tek Optimal Cutting Temperature compound (Ted Pella Inc., Redding, CA). Cryo-blocks were sectioned on a Cryostat Leica CM1850 UV (Leica Biosystems Nussloch GmbH, Nussloch, Germany). Staining was performed with primary and secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).