Rehydration of freeze substituted brain tissue for preembedding immuno-electron microscopy

Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.

Morphological Alterations and Increased S100B Expression in Epidermal Langerhans Cells Detected in Skin from Patients with Progressive Vitiligo

The role of Langerhans cells (LCs) in vitiligo pathogenesis remains unclear, with published studies reporting contradictory results regarding the quantity of LCs and no data on the features of LCs in vitiligo. Here, we aimed to analyze the presence, density, and morphological features of LCs in the epidermis of patients with vitiligo. Skin biopsies were stained for LCs using anti-CD1a/anti-langerin antibodies and analyzed by immunocytochemistry with light and electron microscopy. Compared with healthy controls, we detected significantly increased numbers of epidermal LCs in lesional skin from vitiligo in the progressive state. These LCs exhibited striking morphological alterations, including an elevated number of dendrites, with increased length and more branches than dendrites from controls. Ultrastructure examination via immuno-electron microscopy revealed markedly reduced Birbeck granules (BGs) and shorter BG rods in LCs from progressive vitiligo, with higher expression of langerin. Additionally, expression of S100B, the activity biomarker of vitiligo, was increased in these LCs. This work provides new insight on the cellular composition of LCs in vitiliginous skin, revealing altered morphology and increased LC numbers, with elevated S100B expression. Our data suggest LCs might play a critical role in vitiligo pathogenesis and thus may represent a novel therapeutic target for this disease.

Morpho-functional characterization of the endo-lysosomal system by high-throughput correlative light-electron microscopy

Rab5, EEA1 and APPL1 are frequently used in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as marker for late endosomes and lysosomes. However, since these proteins localize poorly in immuno-electron microscopy, systematic studies on their ultrastructural distributions are lacking. Here we address this gap by presenting a quantitative, high-throughput, on-section correlative light-electron microscopy (CLEM) approach using the sensitivity of fluorescence microscopy to infer label to hundreds of organelles classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 co-localizes with Rab5 on especially early endosomes, but unexpectedly also labels Rab5-negative late endosomes and even lysosomes. APPL1 is restricted to small Rab5-positive, vesicular profiles without any visible content or ultrastructural marks. Rab7 primarily labels late endosomes and lysosomes. Our studies reveal the first ultrastructural distribution of key endosomal proteins at their endogenous levels and introduce CLEM as sensitive alternative for quantitative immuno-EM.

Hherisomes, hedgehog specialized recycling endosomes, are required for high level hedgehog signaling and tissue growth

In metazoans, tissue growth and patterning is partly controlled by the Hedgehog (Hh) morphogen. Using immuno-electron microscopy on Drosophila wing imaginal discs, we identified a cellular structure, the Hherisomes that contain the majority of intracellular Hh. Hherisomes are recycling tubular endosomes and their formation is specifically boosted by overexpression of Hh. Expression of Rab11, a small GTPase involved in recycling endosomes, boosts the size of Hherisomes and their Hh concentration. Conversely, increased expression of the transporter Dispatched, a regulator of Hh secretion, leads to their clearance. We show that increasing Hh density in Hherisomes through Rab11 overexpression enhances both the level of Hh-signaling and disc pouch growth, whereas Dispatched overexpression decreases high level Hh-signaling and growth. We propose that upon secretion, a pool of Hh triggers low level signaling, whereas a second pool of Hh is endocytosed and recycled through Hherisomes to stimulate high level signaling and disc pouch growth. Altogether our data indicate that Hherisomes are required to sustain physiological Hh activity necessary for patterning and tissue growth in the wing disc.

Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy

Immuno-electron microscopy (Immuno-EM) is a powerful tool for identifying molecular targets with ultrastructural details in biological specimens. However, technical barriers, such as the loss of ultrastructural integrity, the decrease in antigenicity, or artifacts in the handling process, hinder the widespread use of the technique by biomedical researchers. We developed a method to overcome such challenges by combining light and electron microscopy with immunolabeling based on Tokuyasu’s method. Using cryo-sectioned biological specimens, target proteins with excellent antigenicity were first immunolabeled for confocal analysis, and then the same tissue sections were further processed for electron microscopy, which provided a well-preserved ultrastructure comparable to that obtained using conventional electron microscopy. Moreover, this method does not require specifically designed correlative light and electron microscopy (CLEM) devices but rather employs conventional confocal and electron microscopes; therefore, it can be easily applied in many biomedical studies.

Proteome Profile of Myelin in the Zebrafish Brain

The velocity of nerve conduction along vertebrate axons depends on their ensheathment with myelin. Myelin membranes comprise specialized proteins well characterized in mice. Much less is known about the protein composition of myelin in non-mammalian species. Here, we assess the proteome of myelin biochemically purified from the brains of adult zebrafish (Danio rerio), considering its increasing popularity as model organism for myelin biology. Combining gel-based and gel-free proteomic approaches, we identified > 1,000 proteins in purified zebrafish myelin, including all known constituents. By mass spectrometric quantification, the predominant Ig-CAM myelin protein zero (MPZ/P0), myelin basic protein (MBP), and the short-chain dehydrogenase 36K constitute 12%, 8%, and 6% of the total myelin protein, respectively. Comparison with previously established mRNA-abundance profiles shows that expression of many myelin-related transcripts coincides with the maturation of zebrafish oligodendrocytes. Zebrafish myelin comprises several proteins that are not present in mice, including 36K, CLDNK, and ZWI. However, a surprisingly large number of ortholog proteins is present in myelin of both species, indicating partial evolutionary preservation of its constituents. Yet, the relative abundance of CNS myelin proteins can differ markedly as exemplified by the complement inhibitor CD59 that constitutes 5% of the total zebrafish myelin protein but is a low-abundant myelin component in mice. Using novel transgenic reporter constructs and cryo-immuno electron microscopy, we confirm the incorporation of CD59 into myelin sheaths. These data provide the first proteome resource of zebrafish CNS myelin and demonstrate both similarities and heterogeneity of myelin composition between teleost fish and rodents.

The AL626 antibody recognizes an ALFA-tagged recombinant protein by immuno-electron microscopy

The AL626 antibody against the ALFA tag recognizes an ALFA-tagged human CD1b protein by immuno-electron microscopy in transfected HEK293T cells.