Resin comparison for serial block face scanning volume electron microscopy.

Serial Block Face Scanning Electron Microscopy (SBF-SEM) is one of several volume electron microscopy (vEM) techniques whose purpose is to reveal the nanostructure of cells and tissues in three dimensions. As one of the earliest, and possibly most widely adopted of the disruptive vEM techniques there have been hundreds of publications using the method, although very few comparative studies of specimen preparation parameters. While some studies have focused on staining and specimen acquisition no comparison of resin embedding has yet been conducted.

Light triggered nanoscale biolistics for efficient intracellular delivery of functional macromolecules in mammalian cells.

Biolistic intracellular delivery of functional macromolecules makes use of dense microparticles which are ballistically fired onto cells with a pressurized gun. While it has been used to transfect plant cells, its application to mammalian cells has met with limited success mainly due to high toxicity. Here we present a more refined nanotechnological approach to biolistic delivery with light-triggered self-assembled nanobombs (NBs) that consist of a photothermal core particle surrounded by smaller nanoprojectiles.

Involvement of the Choroid Plexus in the Pathogenesis of Niemann-Pick Disease Type C.

Niemann-Pick type C (NPC) disease, sometimes called childhood Alzheimer's, is a rare neurovisceral lipid storage disease with progressive neurodegeneration leading to premature death. The disease is caused by loss-of-function mutations in the or gene which both result into lipid accumulation in the late endosomes and lysosomes. Since the disease presents with a broad heterogenous clinical spectrum, the involved disease mechanisms are still incompletely understood and this hampers finding an effective treatment.

A workflow for 3D-CLEM investigating liver tissue.

Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient.

Osteopontin Expression Identifies a Subset of Recruited Macrophages Distinct from Kupffer Cells in the Fatty Liver.

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow.

Three-Dimensional Visualization of APEX2-Tagged Erg11 in Saccharomyces cerevisiae Using Focused Ion Beam Scanning Electron Microscopy.

The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3'-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images.

Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche.

Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity.

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy.

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible.

Combining serial block face and focused ion beam scanning electron microscopy for 3D studies of rare events.

Volume electron microscopy allows for the automated acquisition of serial-section imaging data that can be reconstructed in three-dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. Two, volume electron microscopy (EM) techniques, serial block face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), use a similar slice-and-view approach but differ in their fields of view and 3D resolution. This chapter highlights a workflow where the ability of SBF-SEM to image a large field of view is combined with the precise sectioning capability of FIB-SEM to first locate a rare cellular event in a large tissue volume and then inspect the event with higher resolution.

Serial block face-scanning electron microscopy for volume electron microscopy.

There are different technologies that can be used to obtain a 3D image at nanometer resolution. Over the past decade, there has been a growing interest in applying Serial Block Face Scanning Electron Microscopy (SBF-SEM) in different fields of life science research. This technology has the advantage that it can cover a range of volumes, going from monolayers to multiple tissue layers in all three dimensions.

Longitudinal assessment of cerebral perfusion and vascular response to hypoventilation in a bigenic mouse model of Alzheimer's disease with amyloid and tau pathology.

Alzheimer's disease is the most common neurodegenerative disease, and many patients also present with vascular dysfunction. In this study, we aimed to assess cerebral blood flow (CBF) and cerebrovascular response (CVR) as early, pre-symptomatic (3 months of age), imaging markers in a bigenic model of Alzheimer's disease (APP.V717IxTau.

Novel Phospho-Tau Monoclonal Antibody Generated Using a Liposomal Vaccine, with Enhanced Recognition of a Conformational Tauopathy Epitope.

The microtubule-associated protein Tau is an intrinsically unfolded, very soluble neuronal protein. Under still unknown circumstances, Tau protein forms soluble oligomers and insoluble aggregates that are closely linked to the cause and progression of various brain pathologies, including Alzheimer's disease. Previously we reported the development of liposome-based vaccines and their efficacy and safety in preclinical mouse models for tauopathy.

Positive and negative early life experiences differentially modulate long term survival and amyloid protein levels in a mouse model of Alzheimer's disease.

Stress has been implicated as a risk factor for the severity and progression of sporadic Alzheimer's disease (AD). Early life experiences determine stress responsivity in later life, and modulate age-dependent cognitive decline. Therefore, we examined whether early life experiences influence AD outcome in a bigenic mouse model which progressively develops combined tau and amyloid pathology (biAT mice).

Terminal hypothermic Tau.P301L mice have increased Tau phosphorylation independently of glycogen synthase kinase 3α/β.

The microtubule-associated protein Tau is responsible for a large group of neurodegenerative disorders, known as tauopathies, including Alzheimer's disease. Tauopathy result from augmented and/or aberrant phosphorylation of Tau. Besides aging and various genetic and epigenetic defects that remain largely unknown, an important non-genetic agent that contributes is hypothermia, eventually caused by anesthesia.

Phosphorylation of protein Tau by GSK3β prolongs survival of bigenic Tau.P301L×GSK3β mice by delaying brainstem tauopathy.

Tau.P301L transgenic mice suffer precocious mortality between ages 8 and 11 months, resulting from upper airway defects caused by tauopathy in autonomic brainstem circuits that control breathing (Dutschmann et al., 2010).

Early structural and functional defects in synapses and myelinated axons in stratum lacunosum moleculare in two preclinical models for tauopathy.

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer's disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.

Increasing brain protein O-GlcNAc-ylation mitigates breathing defects and mortality of Tau.P301L mice.

The microtubule associated protein tau causes primary and secondary tauopathies by unknown molecular mechanisms. Post-translational O-GlcNAc-ylation of brain proteins was demonstrated here to be beneficial for Tau.P301L mice by pharmacological inhibition of O-GlcNAc-ase.

Efficacy and safety of a liposome-based vaccine against protein Tau, assessed in tau.P301L mice that model tauopathy.

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimer's disease. The unmet need of effective therapy for Alzheimer's disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404.

Neurological characterization of mice deficient in GSK3α highlight pleiotropic physiological functions in cognition and pathological activity as Tau kinase.

Background: GSK3β is involved in a wide range of physiological functions, and is presumed to act in the pathogenesis of neurological diseases, from bipolar disorder to Alzheimer's disease (AD). In contrast, the GSK3α isozyme remained largely ignored with respect to both aspects.

Results: We generated and characterized two mouse strains with neuron-specific or with total GSK3α deficiency.

Tauopathy differentially affects cell adhesion molecules in mouse brain: early down-regulation of nectin-3 in stratum lacunosum moleculare.

Cell adhesion molecules are important structural substrates, required for synaptic plasticity and synaptogenesis. CAMs differ widely in their expression throughout different brain regions and their specific structural and functional roles in the brain remain to be elucidated. Here, we investigated selected cell adhesion molecules for alterations in expression levels and neuronal localization in validated mouse models for Alzheimer's disease that mimic the age-related progression of amyloid accumulation and tauopathy.

Isoflurane anesthesia precipitates tauopathy and upper airways dysfunction in pre-symptomatic Tau.P301L mice: possible implication for neurodegenerative diseases.

The postoperative cognitive decline resulting from volatile anesthesia is gaining acceptance as a major health problem. The common anesthetic isoflurane is suspected to precipitate neurodegeneration in Alzheimer's disease by unknown mechanisms. We previously validated that 8month old Tau.

Early improved and late defective cognition is reflected by dendritic spines in Tau.P301L mice.

Cognitive demise correlates with progressive brain tauopathy in dementing patients. Improved cognition of young Tau.P301L mice contrasts with dysfunction later in life and remains unexplained (Boekhoorn et al.

GSK-3α/β kinases and amyloid production in vivo.

Arising from C. J. Phiel, C.