细胞生物学

Single-cell RNA sequencing has revolutionized molecular cell biology by enabling the identification of unique transcription profiles and cell transcription states within the same tissue. However, tissue dissociation presents a challenge for non-model organisms, as commercial kits are often incompatible, and current protocols rely on tissue enzymatic digestion for extended periods. Tissue digestion can alter cell transcription in response to temperature and the stress caused by enzymatic treatment. Here, we propose a protocol to stabilize RNA using a deep eutectic solvent (Vivophix, Rapid Labs) prior to tissue dissociation, thereby avoiding transcription changes induced by the process and preventing RNase activity during incubation. We validated this methodology for three medically important insect vectors: Anopheles gambiae, Aedes aegypti, and Lutzomyia longipalpis. Single-cell RNA sequencing using our insect midgut dissociation protocol yielded high-quality sequencing results, with a high number of cells recovered, a low percentage of mitochondrial reads, and a low percentage of ambient RNA—two hallmark standards of cell quality.

Fuchs endothelial corneal dystrophy (FECD) is a rare and multifactorial disorder leading to cell death in the innermost layer of the cornea, i.e., the endothelium; UV radiation is reported as the major environmental risk for the disease. Establishing an animal model for this disease has remained challenging in FECD research. We have developed a detailed protocol for the establishment of a UVA-induced FECD mouse model and removal of corneal endothelium from the eye for further molecular and histological studies by taking references from previous studies. UVA light of 500 J/cm² was focused on the C57BL/6J female mouse cornea and kept for an observation period of 90 days. The animal developed corneal scarring by the end of three months. Slit-lamp microscopy and alizarin red–trypan blue staining confirmed endothelial cell death and formation of corneal guttae in the endothelium. Surgical removal of the endothelial layer was successfully done in the diseased mouse, and the result was confirmed by immunofluorescence. This study is relevant for in-depth research using a FECD mouse model, which will surpass the limitation of human tissue scarcity and can be used for in vivo drug targeting to develop therapeutics to cure FECD.

The organ of Corti, located in the inner ear, is the primary organ responsible for animal hearing. Each hair cell has a V-shaped or U-shaped hair bundle composed of actin-filled stereocilia and a kinocilium supported by true transport microtubules. Damage to these structures due to noise exposure, drug toxicity, aging, or environmental factors can lead to hearing loss and other disorders. The challenge when examining auditory organs is their location within the bony labyrinth and their small and fragile nature. This protocol describes the dissection procedure for the cochlear organ, followed by confocal imaging of immunostained endogenous and fluorescent proteins. This approach can be used to understand hair cell physiology and the molecular mechanisms required for normal hearing.

In mammals, the skin comprises several distinct cell populations that are organized into the following layers: epidermis (stratum corneum, stratum granulosum, stratum spinosum, and basal layer), basement membrane, dermis, and hypodermal (subcutaneous fat) layers. It is vital to identify the exact location and function of proteins in different skin layers. Laser capture microdissection (LCM) is an effective technique for obtaining pure cell populations from complex tissue sections for disease-specific genomic and proteomic analysis. In this study, we used LCM to isolate different skin layers, constructed a stratified developmental lineage proteome map of human skin that incorporates spatial protein distribution, and obtained new insights into the role of extracellular matrix (ECM) on stem cell regulation.

The retina is a thin neuronal multilayer responsible for the detection of visual information. The first step in visual transduction occurs in the photoreceptor outer segment. The studies on photoreception and visual biochemistry have often utilized rod outer segments (OS) or OS disks purified from mammalian eyes. Literature reports several OS and disk purification procedures that rarely specify the procedure utilized to collect the retina from the eye. Some reports suggest the use of scissors, while others do not mention the issue as they declare to utilize frozen retinas. Because the OS are deeply embedded in the retinal pigmented epithelium (RPE), the detachment of the retina by a harsh pull-out can cause the fracture of the photoreceptor cilium. Here, we present a protocol maximizing OS yield. Eye semi-cups, obtained by hemisecting the eyeball and discarding the anterior chamber structures and the vitreous, are filled with Mammalian Ringer. After 10–15 min of incubation, the retinas spontaneously detach with their wealth of OS almost intact. The impressive ability of the present protocol to minimize the number of OS stuck inside the RPE, and therefore lost, compared with the classic procedure, is shown by confocal laser scanning microscopy analysis of samples stained ex vivo with a dye (MitoTracker deep red) that stains both retinal mitochondria and OS. Total protein assay of OS disks purified by either procedure also shows a 300% total protein yield improvement. The advantage of the protocol presented is its higher yield of photoreceptor OS for subsequent purification procedures, while maintaining the physiological features of the retina.

The vestibular sensory apparatus contained in the inner ear is a marvelous evolutionary adaptation for sensing movement in 3 dimensions and is essential for an animal’s sense of orientation in space, head movement, and balance. Damage to these systems through injury or disease can lead to vertigo, Meniere’s disease, and other disorders that are profoundly debilitating. One challenge in studying vestibular organs is their location within the boney inner ear and their small size, especially in mice, which have become an advantageous mammalian model. This protocol describes the dissection procedure of the five vestibular organs from the inner ear of adult mice, followed by immunohistochemical labeling of a whole mount preparation using antibodies to label endogenous proteins such as calretinin to label Type I hair cells or to amplify genetically expressed fluorescent proteins for confocal microscopic imaging. Using typical lab equipment and reagents, a patient technician, student, or postdoc can learn to dissect and immunolabel mouse vestibular organs to investigate their structure in health and disease.

The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding how dysregulation can lead to breast cancer initiation and progression. Three-dimensional (3D) mammary organoids offer many exciting possibilities for the study of tissue development and breast cancer. In the present protocol derived from Sumbal et al., we describe a straightforward 3D organoid system for the study of lactation and involution ex vivo. We use primary and passaged mouse mammary organoids stimulated with fibroblast growth factor 2 (FGF2) and prolactin to model the three cycles of mouse mammary gland lactation and involution processes. This 3D organoid model represents a valuable tool to study late postnatal mammary gland development and breast cancer, in particular postpartum-associated breast cancer.

Graphic abstract:

Mammary gland organoid isolation and culture procedures

Microdissection techniques are very important for anatomical and functional studies focused on neuroscience, where it is often necessary microdissect specific brain areas to perform molecular or anatomical analyses. The parafilm^®-assisted microdissection (PAM) was previously described and involves the microdissection of tissue sections mounted on parafilm-covered glass slides. In this work, we describe the use of the PAM method to microdissect rodent nucleus accumbens (NAc). (1) We first describe the best way to perform the mouse euthanasia and how to remove the brain. (2) Next, we describe how to prepare the slides with parafilm^® that will be used to receive the brain slices. (3) Following, we describe how to handle the brain in the cryostat, how to align the hemispheres and how to identify the NAc antero-posterior limits. (4) We also describe how to perform the staining and dehydration of the slices, a critical step to facilitate the microdissection and preserve macromolecules. (5) In the final step, we describe how to identify the dorso-ventral and latero-medial limits of the NAc and, finally, how to perform the manual microdissection of the area. This is a low-cost technique that allows the researcher to specifically microdissect any brain region, from which intact RNA and proteins can be extracted to perform several molecular analyses (e.g., real-time PCR, Western blot, and RNA-seq).

Single cell RNA sequencing is a very powerful means for cellular heterogeneous studies and so becoming wildly utilized nowadays. To guarantee the success of such analysis, it is very important, though sometimes difficult, to obtain single cells suspension with high quality, especially from the primary solid organs like mammary glands. Digestion of mouse or human mammary glands with enzymes was previously described. However, the yield, viability, especially the separation degree of the cells have rarely been noticed in these studies. Here we described a detailed protocol for the single epithelial cells suspension preparation from mouse mammary glands, which could be applied for single cell RNA sequencing on different platforms. This protocol could be well adapted for dissociation of other solid organs and tumors, and the single cell suspension could be also used for many other experiments.

Acute cerebellar slices are widely used among neuroscientists to study the properties of excitatory and inhibitory synaptic transmission as well as intracellular signaling pathways involved in their regulation in cerebellum. The cerebellar cortex presents a well-organized circuitry, and several neuronal pathways can be stimulated and recorded reliably in acute cerebellar slices. A widely used acute cerebellar slice preparation technique was adapted from Edwards’ thin slice preparation method published in 1989 (Edwards et al., 1989). Most of the acute cerebellar slice preparation techniques use a vibrating microtome for slicing freshly dissected cerebellum from various animal species. Here we introduce a simpler method, which uses a tissue chopper to quickly prepare acute sagittal cerebellar slices from rodents. Cerebellum is dissected from the whole brain and sliced with a tissue chopper into 200-400 µm thick slices. Slices are allowed to recover in oxygenated aCSF at 37 °C for 1-2 h. Slices can then be used for electrophysiology or other types of experimentation. This method can be used to prepare cerebellar slices from mouse or rat aged from postnatal day 7 to 2 years. The preparation is faster and easier than other methods and provides a more versatile diversity of applications.