Combining high‐pressure freezing with pre‐embedding immunogold electron microscopy and tomography

Abstract

Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.

Biogenesis of lysosome‐related organelles complex‐1 (BORC) regulates late endosomal/lysosomal size through PIKfyve‐dependent phosphatidylinositol‐3,5‐bisphosphate

“…Specimen preparation, immunoelectron microscopy and electron tomography were performed as previously described in detail . Briefly, for morphology, the cells were cultured on sapphire discs suitable for high‐pressure freezing, freeze substitution and epoxy resin embedding.…”

“…Thin sections (80 nm) were used for standard TEM, and 300‐400 nm‐semi‐thick sections for dual‐tilt scanning transmission electron tomography (STEM, with a TECNAI T20G2 from FEI, now: Thermo Scientific, Waltham, MA, USA). For immunogold EM, we used Tokuyasu‐cryosection labeling or our recently described method for cryobased pre‐embedding immunogold labeling . Primary antibodies were mouse antihuman LAMP1 (EM: 1:10; H4A3; Developmental Studies Hybridoma Bank), rabbit anticathepsin D (EM: 1:100; Ab‐2; Calbiochem), mouse anti‐HA (1:30‐1:100; MMS‐101R; Covance), secondary antibodies were NANOGOLD‐Fab’ goat antimouse and NANOGOLD‐Fab' goat antirabbit IgG (H + L) (1:150; #2002, 2004, respectively, both from Nanoprobes), goat antimouse IgG 5 nm gold (#EM.GAM5, British Biocell) and visualized by silver enhancement (SE) with HQ‐Silver (#2012, Nanoprobes).…”

Research Techniques Made Simple: Cell Biology Methods for the Analysis of Pigmentation

“…A specific area of a thick EM section (250e350 nm) is imaged at incremental angles, and the images are aligned using embedded gold particles as fiduciary marks. ET is preferentially combined with HPF and compatible with IEM (Hess et al, 2018). An alternative and rapidly advancing (but lower resolution) 3D approach is focused ion beam scanning EM, in which a sample is repeatedly imaged on a scanning electron microscope after sequentially removing thin surface layers with a focused ion beam (Titze and Genoud, 2016).…”

Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging

“…Another solution for this issue would be the use of high-pressure freezing and freeze-substitution followed by rehydration, immunostaining using antibodies in the aqueous milieu, and finally embedding and processing for imaging. This approach has been used for immuno-EM in the past (42)(43)(44), and should be applicable also for CLEM procedures based on antibody immunostaining. The nanoSIMS implementation of the CLEM-SIMS procedure is relatively straightforward.…”