Cell Biology

The mammary gland undergoes functional, developmental, and structural changes that are essential for lactation and reproductive processes. An overview of such unique tissue can offer clearer insights into mammary development and tumorigenesis. Compared to traditional methods, mouse mammary gland whole mount is a pivotal technique that provides three-dimensional structural perspectives on gland morphology and developmental stages, offering an inexpensive and accessible approach. This protocol outlines the tissue isolation of the mouse mammary gland and provides detailed instructions for whole-mount staining and analysis. Mammary gland tissues are carefully dissected from euthanized mice and stained with Carmine Alum to highlight the ductal structures, enabling detailed visualization of the branching patterns and morphological changes. Light microscopy is used to capture a panoramic image of the stained mammary gland, enabling the quantitative analysis of terminal end buds (TEBs) and bifurcated TEBs to further investigate mammary gland remodeling. This method can provide invaluable insights, particularly in the study of mammary gland morphogenesis and tumorigenesis, underscoring its significance in both basic research and clinical applications.

With the growth of the quantum biology field, the study of magnetic field (MF) effects on biological processes and their potential therapeutic applications has attracted much attention. However, most biologists lack the experience needed to construct an MF exposure apparatus on their own, no consensus standard exists for exposure methods, and protocols for model organisms are sorely lacking. We aim to provide those interested in entering the field with the ability to investigate static MF effects in their own research. This protocol covers how to design, build, calibrate, and operate a static MF exposure chamber (MagShield apparatus), with instructions on how to modify parameters to other specific needs. The MagShield apparatus is constructed of mu-metal (which blocks external MFs), allowing for the generation of experimentally controlled MFs via 3-axial Helmholtz coils. Precise manipulation of static field strengths across a physiologically relevant range is possible: nT hypomagnetic fields, μT to < 1 mT weak MFs, and moderate MFs of several mT. An integrated mu-metal partition enables different control and experimental field strengths to run simultaneously. We demonstrate (with example results) how to use the MagShield apparatus with Xenopus, planarians, and fibroblast/fibrosarcoma cell lines, discussing the modifications needed for cell culture systems; however, the apparatus is easily adaptable to zebrafish, C. elegans, and 3D organoids. The operational methodology provided ensures uniform and reproducible results, affording the means for rigorous examination of static MF effects. Thus, this protocol is a valuable resource for investigators seeking to explore the intricate interplay between MFs and living organisms.

Tissue-engineered constructs combine the mechanical properties of biomaterials with biological agents to serve as scaffolds that direct the wound-healing process and promote tissue regeneration. A limitation to studying wound healing in vivo is that mouse skin contracts to heal rather than exhibiting granulation tissue formation and epithelialization like human skin. Therefore, it became necessary to develop a mouse model to better recapitulate human wound healing. The first splinted excisional wound healing model in mice, described in 2004, utilized silicone splints to prevent skin contracture. This model has been used to test a variety of wound healing strategies; however, to our knowledge, this model has not been adapted to test the effect of implants on wound healing. In our established protocol, circular bilateral excisional wounds are made on the mouse’s dorsum. A circular implant made of porous polyethylene is sutured to the skin within the wound. A thin, donut-shaped silicone splint is secured to the skin surrounding the wound, and a thick, donut-shaped splint is placed on top to tent the wound dressing. Finally, the mouse’s abdomen is wrapped in a bandage and tape to protect the implants. Our protocol offers a significant enhancement to the existing model by enabling the testing of implants for wound healing, as well as using an additional splint that prevents direct contact between the wound dressing and the wound bed. This model can be used to study tissue-engineered implant designs in a relatively low-cost, simple, and high-throughput manner before advancing to larger animal studies.

Current ischemic models strive to replicate ischemia-mediated injury. However, they face challenges such as inadequate reproducibility, difficulties in translating rodent findings to humans, and ethical, financial, and practical constraints that limit the accuracy of extensive research. This study introduces a novel approach to inducing persistent ischemia in 3-day-old chicken embryos using endothelin-1. The protocol targets the right vitelline arteries, validated with Doppler blood flow imaging and molecular biology experiments. This innovative approach facilitates the exploration of oxidative stress, inflammatory responses, cellular death, and potential drug screening suitability utilizing a 3-day-old chicken embryo.

Cultured mammalian cells are a common model system for the study of epithelial biology and mechanics. Epithelia are often considered as pseudo–two dimensional and thus imaged and analyzed with respect to the apical tissue surface. We found that the three-dimensional architecture of epithelial monolayers can vary widely even within small culture wells, and that layers that appear organized in the plane of the tissue can show gross disorganization in the apical-basal plane. Epithelial cell shapes should be analyzed in 3D to understand the architecture and maturity of the cultured tissue to accurately compare between experiments. Here, we present a detailed protocol for the use of our image analysis pipeline, Automated Layer Analysis (ALAn), developed to quantitatively characterize the architecture of cultured epithelial layers. ALAn is based on a set of rules that are applied to the spatial distributions of DNA and actin signals in the apical-basal (depth) dimension of cultured layers obtained from imaging cultured cell layers using a confocal microscope. ALAn facilitates reproducibility across experiments, investigations, and labs, providing users with quantitative, unbiased characterization of epithelial architecture and maturity.

Key features

• This protocol was developed to spatially analyze epithelial monolayers in an automated and unbiased fashion.

• ALAn requires two inputs: the spatial distributions of nuclei and actin in cultured cells obtained using confocal fluorescence microscopy.

• ALAn code is written in Python3 using the Jupyter Notebook interactive format.

• Optimized for use in Marbin-Darby Canine Kidney (MDCK) cells and successfully applied to characterize human MCF-7 mammary gland–derived and Caco-2 colon carcinoma cells.

• This protocol utilizes Imaris software to segment nuclei but may be adapted for an alternative method. ALAn requires the centroid coordinates and volume of nuclei.

Graphical overview

Structural and functional changes in vascular networks play a vital role during development, causing or contributing to the pathophysiology of injury and disease. Current methods to trace and image the vasculature in laboratory settings have proven inconsistent, inaccurate, and labor intensive, lacking the inherent three-dimensional structure of vasculature. Here, we provide a robust and highly reproducible method to image and quantify changes in vascular networks down to the capillary level. The method combines vasculature tracing, tissue clearing, and three-dimensional imaging techniques with vessel segmentation using AI-based convolutional reconstruction to rapidly process large, unsectioned tissue specimens throughout the body with high fidelity. The practicality and scalability of our protocol offer application across various fields of biomedical sciences. Obviating the need for sectioning of samples, this method will expedite qualitative and quantitative analyses of vascular networks. Preparation of the fluorescent gel perfusate takes < 30 min per study. Transcardiac perfusion and vasculature tracing takes approximately 20 min, while dissection of tissue samples ranges from 5 to 15 min depending on the tissue of interest. The tissue clearing protocol takes approximately 24–48 h per whole-tissue sample. Lastly, three-dimensional imaging and analysis can be completed in one day. The entire procedure can be carried out by a competent graduate student or experienced technician.

Key features

• This robust and highly reproducible method allows users to image and quantify changes in vascular networks down to the capillary level.

• Three-dimensional imaging techniques with vessel segmentation enable rapid processing of large, unsectioned tissue specimens throughout the body.

• It takes approximately 2–3 days for sample preparation, three-dimensional imaging, and analysis.

• The user-friendly pipeline can be completed by experienced and non-experienced users.

Graphical overview

Neovascular diseases of the retina, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), are proliferative retinopathies involving the growth of new blood vessels on the retina, which in turn causes impairment and potential loss of vision. A drawback of conventional angiogenesis assays is that they are not representative of the angiogenic processes in the retina. In the retina, the new blood vessels grow (from pre-existing blood vessels) and migrate into a non-perfused region of the eye including the inner limiting membrane of the retina and the vitreous, both of which contribute to vision loss. The Matrigel Duplex Assay (MDA) measures the migration of angiogenic capillaries from a primary Matrigel layer to a secondary Matrigel layer, which resembles the pathological angiogenesis in AMD and DR. The methodology of MDA is comprised of two steps. In the first step, the human retinal microvascular endothelial cells (HRMECs) are mixed with phenol red–containing Matrigel (in a 1:1 ratio) and seeded in the center of an 8-well chamber slide. After 24 h, a second layer of phenol red–free Matrigel is overlaid over the first layer. Over the course of the next 24 h, the HRMECs invade from the primary Matrigel layer to the secondary layer. Subsequently, the angiogenic sprouts are visualized by brightfield phase contrast microscopy and quantified by ImageJ software. The present manuscript measures the angiogenesis-inhibitory activity of the Src kinase inhibitor PP2 in primary HRMECs using the MDA. The MDA may be used for multiple applications like screening anti-angiogenic drugs, measuring the pro-angiogenic activity of growth factors, and elucidating signaling pathways underlying retinal angiogenesis in normal and disease states.

Graphical overview

Whole-brain clearing and imaging methods are becoming more common in mice but have yet to become standard in rats, at least partially due to inadequate clearing from most available protocols. Here, we build on recent mouse-tissue clearing and light-sheet imaging methods and develop and adapt them to rats. We first used cleared rat brains to create an open-source, 3D rat atlas at 25 μ resolution. We then registered and imported other existing labeled volumes and made all of the code and data available for the community (https://github.com/emilyjanedennis/PRA) to further enable modern, whole-brain neuroscience in the rat.

Key features

• This protocol adapts iDISCO (Renier et al., 2014) and uDISCO (Pan et al., 2016) tissue-clearing techniques to consistently clear rat brains.

• This protocol also decreases the number of working hours per day to fit in an 8 workday.

Graphical overview

Tension and force propagation play a central role in tissue morphogenesis, as they enable sub- and supra-cellular shape changes required for the generation of new structures. Force is often generated by the cytoskeleton, which forms complex meshworks that reach cell–cell or cell–extracellular matrix junctions to induce cellular rearrangements. These mechanical properties can be measured through laser microdissection, which concentrates energy in the tissue of interest, disrupting its cytoskeleton. If the tissue is undergoing tension, this cut will induce a recoil in the surrounding regions of the cut. This protocol describes how one can perform laser microdissection experiments and subsequently measure the recoil speed of the sample of interest. While we explain how to carry out these experiments in Drosophila embryos, the recoil calibration and downstream analyses can be applied to other types of preparations.

Key features

• Allows measuring tension in live Drosophila embryos with a relatively simple approach.

• Describes a quick way to mount a high number of embryos.

• Includes a segmentation-free recoil quantification that reduces bias and speeds up analysis.

Graphical overview

The trachea tube is the exclusive route to allow gas exchange between the external environment and the lungs. Recent studies have shown the critical role of mesenchymal cells in tracheal tubulogenesis. Improved methods for studying the dynamics of the tracheal mesenchyme development are needed to investigate the cellular and molecular mechanisms during tracheal tubulogenesis. Here, we describe a detailed protocol for a systematic analysis of tracheal tube development to enable observing tracheal smooth muscle (SM) and cartilage ring formation. We describe immunostaining, confocal and stereomicroscopy imaging, and quantitative methods to study the process of tracheal SM and cartilage ring development, including SM cell alignment, polarization, and changes in cell shape as well as mesenchymal condensation. The technologies and approaches described here not only improve analysis of the patterning of the developing trachea but also help uncover the mechanisms underlying airway disease. This protocol also provides a useful technique to analyze cell organization, polarity, and nuclear shape in other organ systems.