Cell Biology

Small fiber neuropathy (SFN) is an underdiagnosed condition characterized by sensory and autonomic dysfunction due to impairment of small nerve fibers in skin, blood vessels, and internal organs. Various underlying disorders are associated with SFN, and the pathophysiology of nerve fiber damage and functional impairment is the subject of extensive research. Diagnosis of SFN is challenging as standard electrodiagnostic techniques assess large fiber function and therefore are normal in SFN patients. The current gold standard for SFN diagnosis in humans is a skin biopsy, commonly obtained from the distal leg, hairy skin region, with evaluation of intraepidermal nerve fiber density (IENFD) using protein gene product 9.5 (PGP9.5) immunolabeling. While well-established in clinical practice, equivalent standardized, reproducible methods for assessing IENFD in experimental mouse models are lacking, which limits translational research in this field. Previous work in mice has relied on diverse antibodies, variable tissue sampling, and the use of confocal microscopy to trace nerve fibers. Other approaches have used chromogenic precipitate-based staining, which limits the ability to co-label multiple proteins. Here, we present a detailed, simple, and reproducible protocol for IENFD quantification of small nerves in the distal glabrous skin of the mouse hind paw. This protocol uses the two distal footpads, ensuring consistent sampling across animals. Prior to sectioning, the tissue is fixed and cryoprotected. Serial 20-μm sections are mounted on glass slides, dried, permeabilized, blocked, and immunostained with an anti-PGP9.5 monoclonal antibody, and then detected by binding secondary fluorescent-labeled antibodies. Although murine hairy skin analysis may apparently show a higher translational value, as it better reflects human biopsy sites, it is compromised by dense hair shafts and follicles, which interrupt epidermis continuity and thus interfere with sampling consistency. Polyneuropathy sensory symptoms, in fact, begin at the most distal sensory site, which is the glabrous skin of the toes. Thus, evaluation of this anatomical location best represents the clinical realm and may have the best sensitivity for identifying early axonal changes. In this protocol, we focused on IENFD quantification as done in human samples. Mechanoreceptors such as Meissner corpuscles are detectable and quantifiable by this method, and represent additional value since pressure-evoked pain, transmitted by these, is often reported by affected individuals. This immunolabeling protocol can be completed within one day [involving a small number of animals, where all three stages can be performed during a long working day (approximately 12 h)], while the entire workflow, including fixation and cryoprotection, is completed in up to 72 h. Importantly, the dermal and epidermal small fibers can be visualized using a standard fluorescence microscope, thereby avoiding the need for confocal imaging while maintaining high reproducibility. Preliminary validation in several animal models of inflammatory neuropathy and pain demonstrated a reproducible approximately 50% reduction in IENFD compared to controls, reaching statistical significance with n = 4 per group. This method supports SFN research and preclinical evaluation of novel therapeutics.

Immunohistochemistry (IHC) and immunofluorescence (IF) are fundamental molecular biology techniques to assess protein expression. However, the melanin present normally in the eye in the uveal tract (choroid, iris, and ciliary body) and the retinal pigment epithelium (RPE) poses a significant challenge for IHC and IF. This is because melanin interferes with both chromogenic and fluorescent detection methods. Additionally, formalin fixation, which is commonly used for IHC, can result in shrinkage and loss of cellular detail in the eye. This protocol provides an optimized approach using Davidson’s fixative with a hydrogen peroxide bleaching step to eliminate melanin interference in the mouse eye, improving the quality and interpretability of IHC analyses of the uveal tract and RPE. It is particularly useful for the analysis of uveal melanoma.

Accurate labeling of excitatory postsynaptic sites remains a major challenge for high-resolution imaging due to the dense and sterically restricted environment of the postsynaptic density (PSD). Here, we present a protocol utilizing Sylites, 3 kDa synthetic peptide probes that bind with nanomolar affinity to key postsynaptic markers, PSD-95 and Gephyrin. eSylites (excitatory Sylites) specifically target the PDZ1 and PDZ2 domains of PSD-95, enabling precise and efficient labeling of excitatory postsynaptic density (ePSD). In contrast, iSylites (inhibitory Sylites) bind to the dimerizing E-domain of the Gephyrin C-terminus, allowing selective visualization of inhibitory postsynaptic density (iPSD). Their small size reduces linkage error and enhances accessibility compared to conventional antibodies, enabling clear separation of PSD-95 nanodomains in super-resolution microscopy. The protocol is compatible with co-labeling using standard antibodies and integrates seamlessly into multichannel immunocytochemistry workflows for primary neurons and brain tissue. This method enables robust, reproducible labeling of excitatory synapses with enhanced spatial resolution and can be readily adapted for expansion microscopy or live-cell applications.

The persistence of the HIV-1 reservoir remains the ultimate obstacle in achieving a cure. Cure strategies targeting the HIV-1 reservoir are under development, and therefore, finding ways to improve the detection of the reservoir is crucial. Several reservoir detection techniques exist to assess different markers of the HIV-1 reservoir, such as PCR-based assays and protein-based flow cytometric methods. We developed a flow cytometry-fluorescent in situ hybridization (flow-FISH) approach that assesses HIV-1 at the transcriptional level. Using a combination of probes that target either the HIV-1 trans-activation response (TAR) region and 5′ long terminal repeat (LTR) or the Gag sequence, our assay distinguishes between infected cells expressing abortive or elongated HIV-1 RNAs. This assay utilizes the branched-DNA method to amplify the fluorescent signal of the hybridized RNA probes and can be used directly for thawed or cultured cells, with the option to include surface antibody staining. Cellular expression of abortive and/or Gag HIV-1 RNAs is measured by flow cytometry. Our flow-FISH approach gives insight into the transcriptional dynamics of the HIV-1 reservoir and allows for the characterization of latently infected cells.

One of the major factors contributing to aging and age-related diseases is the well-understood decline in the function of adult stem cells. Quantifying the degree of aging in adult stem cells is essential for advancing anti-aging mechanisms and developing anti-aging agents. However, no systematic approach to this exists. In this study, we developed a method to quantitatively assess the degree of aging in adult intestinal stem cells using a Drosophila midgut model and two aging markers. First, aging was induced in Drosophila with the desired genotype, and the anti-aging agent was administered 7 days before dissection. Then, the levels of two intestinal stem cell aging markers found in Drosophila (PH3 and γ-tubulin) were measured using immunohistochemistry. Finally, fluorescence microscopy was employed to count the number of aging markers and take images, which were analyzed using image analysis software. Using this approach, we quantitatively analyzed the effects of anti-aging agents on the aging of adult intestinal stem cells. This methodology is expected to significantly expedite the development of anti-aging agents and substantially reduce the research costs associated with aging-related studies.

In the intestinal epithelium, intraepithelial lymphocytes (IELs) coexist with intestinal epithelial cells (IECs). The IELs have an important role in defending the intestinal tract against pathogens and eliminating tumor cells. Anomalies in the absolute IEL count have been reported in various digestive diseases. IELs are typically counted using histologic techniques or under light microscopy after isolation of the epithelium. However, these techniques can introduce bias, which might account for the discrepancies in counts from one study to another. Here, we describe a flow cytometry assay for determining the absolute IEL count and the IEL/IEC ratio. We combined a conventional epithelial isolation method with a BD TruCount^TM bead-based absolute counting technique to quantify IELs (CD45⁺ CD326/EpCAM- CD103⁺CD3⁺) and IECs (CD45- CD326/EpCAM⁺) in a C57BL/6 mouse model.

The osteocyte lacuno-canalicular system (LCS) plays a crucial role in maintaining bone homeostasis and mediating cellular mechanotransduction. Current histological techniques, particularly the Ploton silver nitrate staining method, face challenges such as variations in solution concentrations and types as well as a lack of standardization, which limits their broader application in osteocyte research. In this study, we present a simplified and more effective silver nitrate staining protocol designed to address these issues. Our method utilizes a 1 mol/L silver nitrate solution combined with optimized gelatin-formic acid solutions at varying concentrations (0.05%–0.5% type-B gelatin and 0.05%–5% formic acid, or 1%–2% type-B gelatin and 0.1%–2% formic acid). Staining is performed for 1 h under 254 nm ultraviolet light or 90 min under room light, followed by washing with Milli-Q water to terminate staining. This novel optimized method yields consistent and distinct staining of the osteocyte LCS across multiple species, demonstrating superior efficiency and reliability compared to the Ploton method. It will significantly advance research in osteocyte biology and provide a valuable tool for exploring the adaptive evolution of osteocyte LCS morphology and function across various taxa.

Gap junctions are transmembrane protein channels that enable the exchange of small molecules such as ions, second messengers, and metabolites between adjacent cells. Gap junctions are found in various mammalian organs, including skin, endothelium, liver, pancreas, muscle, and central nervous system (CNS). In the CNS, they mediate coupling between neural cells including glial cells, and the resulting panglial networks are vital for brain homeostasis. Tracers of sufficiently small molecular mass can diffuse across gap junctions and are used to visualize the extent of cell-to-cell coupling in situ by delivering them to a single cell through sharp electrodes or patch-clamp micropipettes. Here, we describe a protocol for pre-labeling and identification of astrocytes in acute mouse forebrain slices using Sulforhodamine 101 (SR101). Fluorescent cells can then be targeted for whole-cell patch-clamp, which allows for further confirmation of astroglial identity by assessing their electrophysiological properties, as well as for passive dialysis with a tracer such as biocytin. Slices can then be subjected to chemical fixation and immunostaining to detect dye-coupled networks. This protocol provides a method for the identification of astrocytes in live tissue through SR101 labeling. Alternatively, transgenic reporter mice can also be used to identify astrocytes. While we illustrate the use of this protocol for the study of glial networks in the mouse brain, the general principles are applicable to other species, tissues, and cell types.

Mitochondrial cristae, formed by folding the mitochondrial inner membrane (IM), are essential for cellular energy supply. However, the observation of the IM is challenging due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vitro probes specifically targeting the IM. Here, we describe a detailed imaging protocol for the mitochondrial inner membrane using the Si-rhodamine dye HBmito Crimson, which has excellent photophysical properties, to label live cells for imaging via stimulated emission depletion (STED) microscopy. This allows for STED imaging over more than 500 frames (approximately one hour), with a spatial resolution of 40 nm, enabling the observation of cristae dynamics during various mitochondrial processes. The protocol includes detailed steps for cell staining, image acquisition, image processing, and resolution analysis. Utilizing the superior resolution of STED microscopy, the structure and complex dynamic changes of cristae can be visualized.

Plants use CO₂, water, and light energy to generate carbohydrates through photosynthesis. During daytime, these carbohydrates are polymerized, leading to the accumulation of starch granules in chloroplasts. The catabolites produced by the degradation of these chloroplast starch granules are used for physiological responses and plant growth. Various staining methods, such as iodine staining, have previously been used to visualize the accumulation of chloroplast starch granules; however, these staining methods cannot be used to image live cells and/or provide confocal images with non-specific signals. In this study, we developed a new imaging method for the fluorescent observation of chloroplast starch granules in living plant cells by staining with fluorescein, a widely available fluorescent dye. This simple staining method, which involves soaking a leaf disk in staining solution, shows high specificity in confocal images. Fluorescent images of the stained tissue allow the cellular starch content of living cells to be quantified with the same level of accuracy as a conventional biochemical method (amyloglucosidase/α-amylase method). Fluorescein staining thus not only enables the easy and clear observation of chloroplast starch granules but also allows for precise quantification in living cells.