RESUMO
This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human DC from lymphoid organs, and various non-lymphoid tissues. Within this chapter, detailed protocols are presented that allow for the generation of single-cell suspensions from mouse lymphohematopoietic tissues including spleen, peripheral lymph nodes, and thymus, with a focus on the subsequent analysis of DC by flow cytometry. However, prepared single-cell suspensions can be subjected to other applications including sorting and cellular enrichment procedures, RNA sequencing, Western blotting, and many more. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.
RESUMO
Morphological and molecular characteristics determine the function of biological tissues. Attempts to combine immunofluorescence and electron microscopy invariably compromise the quality of the ultrastructure of tissue sections. We developed NATIVE, a correlated light and electron microscopy approach that preserves ultrastructure while showing the locations of multiple molecular moieties, even deep within tissues. This technique allowed the large-scale 3D reconstruction of a volume of mouse hippocampal CA3 tissue at nanometer resolution.
