Practical Approaches for Cryo-FIB Milling and Applications for Cellular Cryo-Electron Tomography.

Cryo-electron tomography (cryo-ET) is a powerful technique to examine cellular structures as they exist in situ. However, direct imaging by TEM for cryo-ET is limited to specimens up to ∼400 nm in thickness, narrowing its applicability to areas such as cellular projections or small bacteria and viruses. Cryo-focused ion beam (cryo-FIB) milling has emerged in recent years as a method to generate thin specimens from cellular samples in preparation for cryo-ET. In this technique, specimens are thinned with a beam of gallium ions to gradually ablate cellular material in order to leave a thin, electron-transparent section (a lamella) through the bulk material. The lamella can be used for high-resolution cryo-ET to visualize cells in 3D in a near-native state. This approach has proved to be robust and relatively simple for new users and exhibits minimal sectioning artifacts. In this chapter, we describe a general approach to cryo-FIB milling for users with prior cryo-EM experience, with extensive notes on operation and troubleshooting.

Ex vivo kidney slice preparations as a model system to study signaling cascades in kidney epithelial cells.

Several model systems have been used to study signaling cascades in kidney epithelial cells, including kidney histology after systemic treatments, ex vivo isolated tubule perfusion, epithelial cell lines in culture, kidney micropuncture, and ex vivo kidney slices. We and others have found the ex vivo kidney slice method useful to study the signaling cascades involved in the regulation of kidney transport proteins. In this chapter we describe our adaptations to this classic method for the study of the regulation of kinases and endocytosis in rodent kidney epithelial cells. Briefly, slices are obtained by sectioning of freshly harvested rat or mouse kidneys using a Stadie-Riggs tissue slicer. Alternatively, a vibratome can be used to obtain slices at a more consistent and finer thickness. The harvested kidney and kidney slices are kept viable in either cell culture media or in buffers that mimic physiological conditions equilibrated with 5% CO2 at body temperature (37°C). These buffers keep the slices viable during hours for incubations in the presence/absence of different pharmacological agents. After the incubation period the slices can be used for biochemistry experiments by preparing tissue lysates or for histological evaluation after fixation. Moreover, the fixed slices can be used to evaluate changes in subcellular trafficking of epithelial proteins or endosomes via immunolabeling followed by confocal microscopy. The resulting micrographs can then be used for systematic quantification of protein- or compartment-specific changes in subcellular localization under each condition.

Analysis of primary cilia in renal tissue and cells.

Primary cilia are singular, sensory organelles that extend from the plasma membrane of most quiescent mammalian cells. These slender, microtubule-based organelles receive and transduce extracellular cues and regulate signaling pathways. Primary cilia are critical to the development and function of many tissue types, and mutation of ciliary genes causes multi-system disorders, termed ciliopathies. Notably, renal cystic disease is one of the most common clinical features of ciliopathies, highlighting a central role for primary cilia in the kidney. Additionally, acute kidney injury and chronic kidney disease are associated with altered primary cilia lengths on renal epithelial cells, suggesting ciliary dynamics and renal physiology are linked. Here we describe methods to examine primary cilia in kidney tissue and in cultured renal cells. We include immunofluorescence and scanning electron microscopy to determine ciliary localization of proteins and cilia structure. Further, we detail cellular assays to measure cilia assembly and disassembly, which regulate cilia length.

Quantifying autophagic flux in kidney tissue using structured illumination microscopy.

Kidney disease is estimated to affect 15% of the world's population. Autophagy is a key homeostatic pathway in eukaryotic cells, which has been linked to numerous pathological states. In the kidney, autophagy has been shown to modulate both acute and chronic injuries. Despite the importance of autophagy in kidney disease, few techniques to precisely monitor autophagic flux in kidney tissue are available. Here we describe an improved technique to quantify autophagic flux using an RFP-GFP-LC3 reporter mouse and super-resolution microscopy. Using structured illumination microscopy, we can resolve individual autophagosomes within kidney tubular cells. We describe the preparation of slides, staining, imaging and data processing. 3D surface rendering is utilized to categorize and quantify autophagosomes by number, size, fluorescence and autophagic flux in response to ischemia.

A mechanized device for mounting histological tissue sections.

BACKGROUND: Traditional methods for mounting tissue sections onto slides are suboptimal as the amount of labor required quickly multiplies with increasing number of samples. Methods to accelerate the tissue mounting process while reducing the associated risk of tissue damage are needed. NEW METHOD: We designed and 3D printed a mechanized device with an inclined platform used to mount tissue sections onto slides in buffer solution. The main advantage of this design is to reduce the time required for mounting sections as well as minimize the possibility of damaging delicate or thin tissue sections. RESULTS: Using our device, we illustrate and describe in detail the steps required to mount smaller coronally cut mouse brain sections, as well as bigger tangentially cut ferret brain sections. This method's efficiency was assessed by comparing the time required to mount an entire slide of ferret brain sections using our method and the conventional method. Using our device reduced the tissue mounting time by 60%. COMPARISON WITH EXISTING METHOD(S): Compared to existing conventional tissue mounting methods, our device is a simple and user friendly alternative that substantially reduces the time required to mount tissue sections while preserving tissue section quality. CONCLUSIONS: Using our device can streamline histological processing and prove to be especially useful for a variety of tissue types as the platform was designed to accommodate different size microscope slides, and thus use for varying tissue section sizes.

Feline Sporothrix spp. detection using cell blocks from brushings and fine-needle aspirates: Performance and comparisons with culture and histopathology.

BACKGROUND: Sporotrichosis is an emerging zoonotic mycosis that presents as a cutaneous lymphatic or disseminated disease, caused by fungi from the Sporothrix schenkii (S schenkii) clinical clade. Its importance is growing, primarily due to an outbreak that occurred in Brazil, affecting mainly cats and people. OBJECTIVES: In Brazil, an S schenkii diagnosis is often made using cultures, which allows genus identification and sufficient growth to perform molecular biology testing. Despite its advantages, fungal cultures are slow to develop and can delay public health measures, highlighting the importance of developing additional diagnostics techniques. METHODS: Cell block cytology (CBLC) is an older method that regained importance after liquid-based cytology (LBC) was introduced, and it has been previously and successfully applied to veterinary diagnostics. We aimed to standardize and compare CBLC from cervical brush exfoliation of open wounds and fine-needle aspirates with culture and immunohistochemistry of skin biopsies for sporotrichosis in cats, as a novel method. RESULTS: For this purpose, we selected 40 cats with skin lesions suspected of having sporotrichosis in Guarulhos city, São Paulo state, Brazil. We achieved 97.5% and 95% positivity using CBLC and culture, respectively, and 100% of feline skin biopsies were positive for Sporothrix spp on histopathology/immunohistochemistry. CONCLUSIONS: Cell block cytology is an efficient and rapid tool to diagnose sporotrichosis in cats, particularly during epidemics.

Isolation and Generation of Osteoblasts.

This chapter describes the isolation, culture, and staining of osteoblasts. The key advantages of this assay are that it allows direct measurement of bone matrix deposition and mineralization, as well as yielding good quantities of osteoblasts at defined stages of differentiation for molecular and histological analysis. An additional focus of this chapter will be the culture of osteoblasts from less conventional animal species.

Analysis of mRNA, miRNA, and DNA in Bone Cells by RT-qPCR and In Situ Hybridization.

The aim of this chapter is to describe a method used to evaluate gene expression and microRNAs (miRNAs) in bone cells or tissue using Reverse transcription and quantitative Polymerase Chain Reaction (RT-qPCR), and a method to assess chromogenic in situ hybridization (CISH) on Formalin Fixed Paraffin Embedded (FFPE ) mouse bone tissue to detect both DNA and mRNA transcripts using the double digoxigenin (DIG) locked nucleic acid (LNA™) probes .

Osteoarthritis Mouse Model of Destabilization of the Medial Meniscus.

This chapter describes the surgical procedure for destabilization of medial meniscus in mice. Details on subsequent microCT and histological analysis are also provided, as well as details on osteoarthritis evaluation.

Histomorphometry in Rodents.

Bone histomorphometry remains an important tool with which to study the pathophysiology of bone disease and the cellular mechanism by which treatments work. Here we review the methods for embedding, sectioning, staining, and analysis of bone sections in rodents.

Analysis of Bone Architecture in Rodents Using Micro-Computed Tomography.

This chapter describes the use of micro-computed tomography scanning for analyzing bone structure, focussing on rodent bone. It discusses sample preparation, the correct setup of the scanner, the impact of some of the important scanner settings and new applications.

Scanning Electron Microscopy of Bone.

This chapter describes methods for preparing samples of bone and bone cells for scanning electron microscopy (SEM). Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. Samples may have 3D detail in a 3D surface, or be topography-free, polished or micromilled, resin-embedded block surfaces, or resin casts of space compartments surrounded by bone matrix. Methods for cells include fixation, drying, looking at undersides of bone cells, and metallic conductive coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralized matrix is described in detail. Attention is given especially to methods for 3D BSE SEM imaging of bone samples. Recommendations are made for the types of resin embedding for BSE SEM imaging. Correlated confocal and SEM imaging of PMMA embedded bone requires the use of glycerol to coverslip. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualizing fluorescent mineralizing front labels such as calcein and tetracyclines. Making spatial casts from PMMA or other resin-embedded samples is an important use of this material. Correlation with other imaging means, including microradiography and microtomography is important. Shipping wet bone samples between labs is best done in glycerol. Control of the vacuum pressure in the SEM sample chamber (now generally available) can be used to eliminate "charging" problems which were common, for example, with large, complex, cancellous bone samples.

Transmission Electron Microscopy of Bone.

Electron microscopic analysis of mineralized tissues like bone and dentin is essential for understanding of cell-cell/cell-matrix interactions, and the three-dimensional organization of these tissues. This chapter describes a few methods to process mineralized tissues obtained from different sources for ultrastructural analysis by transmission electron microscopy.

Application of Electron Microscopes in Nanotoxicity Assessment.

In this chapter, we highlight the applications of electron microscopes (EMs) in nanotoxicity assessment. EMs can provide detailed information about the size and morphology of nanomaterials (NMs), their localization in cells and tissues, the nano-bio interactions, as well as the ultrastructural changes induced by NMs exposure. Here, we share with the readers how we prepare the tissue sample, and the different types of EMs used among the nanotoxicologists. It is possible to deploy conventional EMs along or in combination with other analytical techniques, such as electron energy loss spectroscopy (EELS), energy dispersive X-ray spectroscopy (EDS or EDX), and TEM-assisted scanning transmission X-ray microscopy (STXM), toward further elemental and chemical characterization. Appropriate images are inserted to illustrate throughout this chapter.

Native System and Cultured Cell Electrophysiology for Investigating Anesthetic Mechanisms.

Anesthetic agents interact with a variety of ion channels and membrane-bound receptors, often at agent-specific binding sites of a single protein. These molecular-level interactions are ultimately responsible for producing the clinically anesthetized state. Between these two scales of effect, anesthetic agents can be studied in terms of how they impact the physiology of neuronal circuits, individual neurons, and cells expressing individual receptor types. The acutely dissected hippocampal slice is one of the most extensively studied and characterized preparations of intact neural tissue and serves as a highly useful experimental model system to test hypotheses of anesthetic mechanisms. Specific agent-receptor interactions and their effect on excitable membranes can further be defined with molecular precision in cell-based expression systems. We highlight several approaches in these respective systems that we have used and that also have been used by many investigators worldwide. We emphasize economy and quality control, to allow an experimenter to carry out these types of studies in a rigorous and efficient manner.

Macroscopic Assessment and Cut Up of Endoscopic Resection Specimens for Early Esophageal Glandular Malignancies.

Pathological assessment of tissue is the gold standard for diagnosis and staging of neoplasia and provides key prognostic information for clinical management. Proper macroscopic assessment and cut-up technique is essential to ensure that the overall assessment is correct and reproducible. Endoscopic mucosal resection is a technique used for removing early neoplastic glandular lesions of the esophagus at the level of submucosa. Here, we describe the macroscopic assessment and dissection techniques used for the routine handling of endoscopic mucosal resection specimens in the clinical laboratory.

Processing of Surgical Specimen (Esophagogastrectomy) for Esophageal Adenocarcinoma.

An esophagogastrectomy is a surgical procedure that is performed for treatment of confirmed localized esophageal and esophagogastric junction adenocarcinoma. Proper macroscopic assessment and cut-up technique is essential to ensure that the overall assessment is correct and reproducible. Here, we describe a standard for macroscopic assessment and dissection to be used for routine handling of esophagogastrectomy specimens in the clinical laboratory.

Quality control in diagnostic immunohistochemistry: integrated on-slide positive controls.

Standardization in immunohistochemistry is a priority in modern pathology and requires strict quality control. Cost containment has also become fundamental and auditing of all procedures must take into account both these principles. Positive controls must be routinely performed so that their positivity guarantees the appropriateness of the immunohistochemical procedure. The aim of this study is to develop a low cost (utilizing a punch biopsy-PB-tool) procedure to construct positive controls which can be integrated in the patient's tissue slide. Sixteen frequently used control blocks were selected and multiple cylindrical samples were obtained using a 5-mm diameter punch biopsy tool, separately re-embedding them in single blocks. For each diagnostic immunoreaction requiring a positive control, an integrated PB-control section (cut from the appropriate PB-control block) was added to the top right corner of the diagnostic slide before immunostaining. This integrated control technique permitted a saving of 4.75% in total direct lab costs and proved to be technically feasible and reliable. Our proposal is easy to perform and within the reach of all pathology labs, requires easily available tools, its application costs is less than using external paired controls and ensures that a specific control for each slide is always available.

Improvements in cell block processing: The Cell-Gel method.

BACKGROUND: The ability to produce adequate cell blocks profoundly impacts the diagnostic usefulness of cytology specimens. Cell blocks are routinely processed from fine-needle aspiration specimens or concentrated fluid samples. Obtaining directed passes for the sole purpose of producing a cell block is common practice, particularly when the cytopathologist anticipates the need for ancillary immunocytochemical stains and/or molecular studies. METHODS: The authors developed an effective and inexpensive process for producing cell blocks that consistently yields abundant cellular material, which they have termed the Cell-Gel method. This method can be simplified into 3 main steps: 1) preparing the sample; 2) constructing the cell block; and 3) processing the cell block. Highlights of the protocol include using a hemolytic fixative for sample preparation and disposable base molds for cell block construction. RESULTS: The cell block failure rate in the current study decreased from 18% with the HistoGel Tube method (January 2014-December 2014) to 6% with the Cell-Gel method (January 2015-December 2016). The authors evaluated 110 cell blocks processed with the HistoGel Tube method and 110 cell blocks processed with the Cell-Gel method, for a total evaluation of 220 cell blocks. CONCLUSIONS: The authors have developed an effective and inexpensive protocol for producing cell blocks that consistently yields abundant cellular material. The Cell-Gel method uses a hemolytic fixative and disposable base molds to produce adequate cell blocks. When the method was implemented, the cell block failure rate of the study laboratory decreased by approximately 67%. Cancer Cytopathol 2017;125:267-276. © 2016 American Cancer Society.