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1.
J Vis Exp ; (167)2021 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-33554959

RESUMO

The morphology, size and quantity of cells, starch granules and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.


Assuntos
Microtomia/métodos , Resinas Sintéticas/química , Sementes/química , Zea mays/química , Proteínas de Plantas/metabolismo , Sementes/citologia , Coloração e Rotulagem , Amido/metabolismo , Zea mays/citologia , Zea mays/embriologia
2.
Methods Mol Biol ; 2223: 87-100, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226589

RESUMO

Allergic contact dermatitis (ACD) is a common skin disease with high prevalence in work environments. Human allergic contact dermatitis is triggered by the exposure to haptens that leads to an initial phase known as sensitization. During this phase, hapten-protein complexes presented by antigen-presenting cells activate a T-cell-mediated response, leading to the generation of memory cells against the hapten. Upon re-exposure to the same hapten, the elicitation phase is initiated. This phase is characterized by a quicker acute inflammatory response involving activation and/or infiltration of a variety of immune cell populations. Human ACD can be studied through the use of animal models of contact hypersensitivity (CHS). The 2,4-dinitrofluorobenzene (DNFB)-induced CHS model is a commonly used mouse model that has been helpful in the study of the mechanisms as well as potential therapeutic interventions of ACD. In this chapter I will provide a detailed protocol to develop acute DNFB-induced CHS in mice in a period of 7 days. In addition, I will discuss several key considerations for experimental design including best controls, potential expected outcomes, and sample collection.


Assuntos
Dermatite Alérgica de Contato/imunologia , Dinitrofluorbenzeno/farmacologia , Modelos Animais de Doenças , Histocitoquímica/métodos , Pele/efeitos dos fármacos , Administração Cutânea , Animais , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/patologia , Orelha , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microtomia , Inclusão em Parafina , Pele/imunologia , Pele/patologia
3.
Methods Mol Biol ; 2223: 217-236, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226598

RESUMO

Cellular inflammation, with elevated levels of Th1/Th2 cytokines, airway mucus hypersecretion, and thickening of the airway smooth muscle, are characteristic features of the allergic lung. Assessment of pathophysiological changes in allergic lungs serves as an important tool to determine disease progression and understand the underlying mechanisms of pathogenesis. This can be achieved by evaluating the lung tissue for inflammation and airway structural changes along with the measurement of important pro-inflammatory mediators such as Th1/Th2 cytokines and eotaxins. This chapter describes procedures to histologically evaluate inflammatory and pathological changes observed during allergic airway inflammation using lung tissue from mice.


Assuntos
Alérgenos/administração & dosagem , Asma/imunologia , Pulmão/imunologia , Hipersensibilidade Respiratória/imunologia , Coloração e Rotulagem/métodos , Equilíbrio Th1-Th2 , Animais , Asma/induzido quimicamente , Asma/metabolismo , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Progressão da Doença , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Microtomia/métodos , Muco/imunologia , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/patologia , Inclusão em Parafina/métodos , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th2/imunologia , Células Th2/patologia
4.
Methods Mol Biol ; 2223: 237-266, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226599

RESUMO

Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin-eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright eosin staining in the secondary granules. For this reason, antibody-mediated detection of eosinophils is preferable for specific and clearer identification of these cells. Moreover, eosinophils may degranulate, releasing their granule proteins into surrounding tissue, and remnants of cytolysed cells cannot be detected by HE staining. The methods here demonstrate the use of eosinophil-specific anti-mouse antibodies to detect eosinophil granule proteins in formalin-fixed cells both in situ in paraffin-embedded lungs, as well as in cytospin preparations from the lung. These antibody staining techniques enable either colorimetric or fluorescence imaging of eosinophils or their granule proteins with the potential for additional antibodies to be added for detection of multiple molecules.


Assuntos
Asma/imunologia , Eosinófilos/imunologia , Imuno-Histoquímica/métodos , Pulmão/imunologia , Hipersensibilidade Respiratória/imunologia , Coloração e Rotulagem/métodos , Alérgenos/administração & dosagem , Animais , Asma/induzido quimicamente , Asma/metabolismo , Asma/patologia , Biomarcadores/metabolismo , Proteína Básica Maior de Eosinófilos/imunologia , Proteína Básica Maior de Eosinófilos/metabolismo , Peroxidase de Eosinófilo/imunologia , Peroxidase de Eosinófilo/metabolismo , Eosinófilos/patologia , Formaldeído/química , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microtomia/métodos , Inclusão em Parafina/métodos , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Fixação de Tecidos/métodos
5.
Methods Mol Biol ; 2223: 267-280, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226600

RESUMO

The intestine is often examined histologically in connection with allergies and in search for pathological changes. To be able to examine the intestine histologically with a microscope, it must be sampled and processed correctly. For microscopic analysis, the samples have to be cut into thin sections, stained, and mounted on slides. Since it is not possible to cut fresh samples without damaging them, they must first be fixed. The most common method, which is described herein, is the fixation in formalin with subsequent embedding in paraffin and staining of the slides with hematoxylin and eosin (H&E). Hematoxylin solutions (in this case Mayer's hemalum solution) stain the acidic components of the cell, i.e., cell nuclei, blue. The staining with eosin gives a pink staining of cytoplasm. This chapter describes the method of processing intestinal tissue for paraffin-embedding, sectioning, and staining with H&E. Tissue processing can be done in tissue processing machines or manually. We describe the manual processing that is often used for smaller batches of samples.


Assuntos
Íleo/patologia , Jejuno/anatomia & histologia , Inclusão em Parafina/métodos , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos , Animais , Galinhas , Amarelo de Eosina-(YS)/química , Formaldeído/química , Hematoxilina/química , Imuno-Histoquímica/métodos , Jejuno/citologia , Microtomia/métodos , Inclusão em Parafina/instrumentação , Suínos , Fixação de Tecidos/instrumentação
6.
Methods Mol Biol ; 2219: 99-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33074536

RESUMO

Trichoplax adhaerens is an enigmatic animal with an extraordinarily simple morphology and a cellular organization, which are the focus of current research. Protocols outlined here provide detailed descriptions of advanced techniques for light and electron microscopic studies of Trichoplax. Studies using these techniques have enhanced our understanding of cell type diversity and function in placozoans and have provided insight into the evolution, development, and physiology of this little understood group.


Assuntos
Microscopia Eletrônica/métodos , Microscopia/métodos , Placozoa/ultraestrutura , Animais , Criopreservação/métodos , Imuno-Histoquímica/métodos , Microtomia/métodos , Placozoa/citologia , Fixação de Tecidos/métodos
7.
Methods Mol Biol ; 2208: 255-264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32856268

RESUMO

Electron microscopy offers necessary precision for the characterization of peptide materials at the nanoscale. Analysis is typically performed for acellular material specimens, whereas measurements in more complex, cellular environments prompt additional considerations for sample processing. Herein, we describe a protocol for the ultramicrotomy analysis of peptide-treated bacterial and mammalian cells. An emphasis is made on cell analysis following peptide treatment, as opposed to peptide analysis in cells, and focuses on sample processing, including fixation and staining procedures, resin embedding, sectioning, and imaging. The application of the protocol is demonstrated for intracellular measurements using antimicrobial peptide materials.


Assuntos
Microtomia/métodos , Peptídeos/farmacologia , Animais , Bactérias/efeitos dos fármacos , Mamíferos , Microscopia Eletrônica/métodos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Coloração e Rotulagem/métodos
8.
Methods Mol Biol ; 2230: 259-281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197019

RESUMO

A method for preparing frozen sections with an adhesive film is described. In order to observe fine structures and weak fluorescence of samples, new types of adhesive films [Cryofilm type 3C(16UF) and 4D(16UF)] are used. The adhesive film is made with very clear and very low autofluorescence. For gene analysis, a very thin adhesive film (LMD film) is used to cut by means of the laser microdissection (LMD). For MALDI mass spectrometry imaging (MALDI-MSI), a conductive adhesive film (Cryofilm type MS) is used to avoid electric charge of the sample. A biological sample is frozen quickly and freeze-embedded. The frozen sample is cut with a very sharp disposable blade made from fine tungsten carbide. The combination of the adhesive films and the blade can generate 3 micrometer thick sections from samples including bone, while it is also possible to generate 1 µm thick sections. The morphology of bone and soft tissues are preserved using this method. Cells such as osteoblasts, fibroblasts, and osteoclasts are clearly observed with an oil immersion lens at high magnification. Sections generated using the Cryofilm type 3C(16UF) shows weak fluorescent signals more clearly than sections generated with the previously reported adhesive films [Cryofilm type 2C(9) and 2C(10)]. Furthermore fluorescence of the fine structures in cells is clearly shown using a super-high-resolution microscope. Several staining and experimental methods such as histology, histochemistry, enzyme histochemistry, immunohistochemistry, and in situ hybridization can be performed on these sections. This method is also useful for preparing frozen sections of large sample such as a whole-body mouse and rat. In gene analysis, gene quality of sample collected from the section made with the LMD film is superior to that of sample made by a conventional method. The Cryofilm type MS makes almost complete section from tissues including hard tissues and large samples. The satisfactory signals are detected from the section with MALDI-MSI.


Assuntos
Osso e Ossos/ultraestrutura , Secções Congeladas/métodos , Histocitoquímica/métodos , Microtomia/métodos , Animais , Criopreservação/métodos , Fibroblastos/ultraestrutura , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Camundongos , Microscopia/métodos , Osteoblastos/ultraestrutura , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
9.
Methods Mol Biol ; 2230: 283-302, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197020

RESUMO

Cartilage and bone are specialized skeletal tissues composed of unique extracellular matrices. Bone, in particular, has a highly calcified or mineralized matrix that makes microtomy and standard histological studies very challenging. Therefore, methods to appropriately fix and decalcify mineralized skeletal tissues have been developed to allow for paraffin processing and standard microtomy. In this chapter, we will illustrate methods for tissue grossing, fixation, decalcification, paraffin processing, embedding, sectioning, and routine histological staining of demineralized murine skeletal tissues. We will also discuss methods for decalcified frozen sectioning of skeletal tissues with and without the use of a tape-transfer system.


Assuntos
Osso e Ossos/ultraestrutura , Cartilagem/ultraestrutura , Técnica de Descalcificação/métodos , Microtomia/métodos , Animais , Secções Congeladas/métodos , Camundongos , Inclusão em Parafina/métodos , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos
10.
J Vis Exp ; (161)2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32716375

RESUMO

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol's critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.


Assuntos
Envelhecimento , Hipocampo/patologia , Hipóxia/patologia , Animais , Hipocampo/fisiologia , Técnicas In Vitro , Camundongos , Microtomia , Neurônios/patologia
11.
J Vis Exp ; (160)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32628156

RESUMO

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.


Assuntos
Cálcio/fisiologia , Técnicas Eletrofisiológicas Cardíacas , Função Ventricular , Ventrículos do Coração , Humanos , Técnicas In Vitro , Potenciais da Membrana , Microtomia
12.
Korean J Radiol ; 21(6): 746-755, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32410413

RESUMO

OBJECTIVE: To identify predictors of pulmonary fibrosis development by combining follow-up thin-section CT findings and clinical features in patients discharged after treatment for COVID-19. MATERIALS AND METHODS: This retrospective study involved 32 confirmed COVID-19 patients who were divided into two groups according to the evidence of fibrosis on their latest follow-up CT imaging. Clinical data and CT imaging features of all the patients in different stages were collected and analyzed for comparison. RESULTS: The latest follow-up CT imaging showed fibrosis in 14 patients (male, 12; female, 2) and no fibrosis in 18 patients (male, 10; female, 8). Compared with the non-fibrosis group, the fibrosis group was older (median age: 54.0 years vs. 37.0 years, p = 0.008), and the median levels of C-reactive protein (53.4 mg/L vs. 10.0 mg/L, p = 0.002) and interleukin-6 (79.7 pg/L vs. 11.2 pg/L, p = 0.04) were also higher. The fibrosis group had a longer-term of hospitalization (19.5 days vs. 10.0 days, p = 0.001), pulsed steroid therapy (11.0 days vs. 5.0 days, p < 0.001), and antiviral therapy (12.0 days vs. 6.5 days, p = 0.012). More patients on the worst-state CT scan had an irregular interface (59.4% vs. 34.4%, p = 0.045) and a parenchymal band (71.9% vs. 28.1%, p < 0.001). On initial CT imaging, the irregular interface (57.1%) and parenchymal band (50.0%) were more common in the fibrosis group. On the worst-state CT imaging, interstitial thickening (78.6%), air bronchogram (57.1%), irregular interface (85.7%), coarse reticular pattern (28.6%), parenchymal band (92.9%), and pleural effusion (42.9%) were more common in the fibrosis group. CONCLUSION: Fibrosis was more likely to develop in patients with severe clinical conditions, especially in patients with high inflammatory indicators. Interstitial thickening, irregular interface, coarse reticular pattern, and parenchymal band manifested in the process of the disease may be predictors of pulmonary fibrosis. Irregular interface and parenchymal band could predict the formation of pulmonary fibrosis early.


Assuntos
Infecções por Coronavirus/diagnóstico por imagem , Pneumonia Viral/diagnóstico por imagem , Fibrose Pulmonar/diagnóstico por imagem , Fibrose Pulmonar/virologia , Adulto , Idoso , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Feminino , Humanos , Masculino , Microtomia/métodos , Pessoa de Meia-Idade , Pandemias , Alta do Paciente , Derrame Pleural/patologia , Derrame Pleural/virologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Valor Preditivo dos Testes , Fibrose Pulmonar/patologia , Estudos Retrospectivos , Tomografia Computadorizada por Raios X/métodos
13.
Int. j. morphol ; 38(2): 389-391, abr. 2020. graf
Artigo em Inglês | LILACS | ID: biblio-1056452

RESUMO

Plastination has revolutionized the study and research of anatomy, thanks to the biosecurity and indefinite preservation of human and animal bodies and organs. This paper presents the concept of Micro-Plastination, an ultra-thin sheet plastination technique, to obtain ultra-thin slices, of a thickness of less than 250 µm, for the identification and visualization of the microanatomy of any anatomical region in morphological and pathological experimental protocols.


La plastinación ha revolucionado el estudio y la investigación de la anatomía, gracias a la conservación biosegura y por tiempo indefinido de cadáveres y órganos humanos y animales. En este trabajo se presenta el concepto de Micro-Plastinación, técnica de plastinación de cortes ultrafinos para la obtención de cortes ultradelgados, de un grosor inferior a los 250 µm, para la identificación y visualización de la microanatomía de cualquier región anatómica en protocolos de morfología experimental.


Assuntos
Humanos , Plastinação/métodos , Anatomia/métodos , Microtomia/métodos
14.
Micron ; 132: 102841, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32062296

RESUMO

The histological study of hard pieces such as tendons and calcified lesions and tissues is a field that has been gaining increased attention owing to the rapid development of implantable prostheses, among other factors. In these studies, serial sectioning is utilized to detect areas of interest throughout the entire piece, as it enables the application of the appropriate light and electron microscopy techniques in these areas. We propose the "three-sectioning method" that subjects the pieces to three consecutive cycles of embedding and sectioning to localize and study the areas of interest, as an efficient technique for these histological studies. The pieces were first embedded in epoxy resin and then cut into thick sections (approximately 300 µm) for the first cycle. Next, areas of interest selected on these thick sections were re-embedded in epoxy resin to be sectioned again (second sectioning) to obtain a series of semithin sections (1-3 µm). These semithin sections are usually studied using the most relevant techniques for light microscopy. Smaller areas of interest are selected to be cut into ultrathin sections (60-90 nm) for transmission electron microscopy. If necessary, the selected areas of the semithin sections can be embedded again, and then cut into new ultrathin sections. The different kinds of sections we have described here may also be studied using scanning electron microscopy. This systematic method facilitates correlative microscopy from lower to higher magnifications along with the usage of a broad variety of histological techniques including electron microscopy.


Assuntos
Técnicas Histológicas/métodos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microtomia/métodos , Manejo de Espécimes/métodos , Animais , Osso e Ossos/ultraestrutura , Resinas Epóxi , Feminino , Masculino , Ratos Wistar , Tendões/ultraestrutura
15.
Molecules ; 25(4)2020 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-32079064

RESUMO

CRANAD-28, a difluoroboron curcumin analogue, has been demonstrated in earlier reports to successfully label amyloid beta (Aß) plaques for imaging both ex vivo and in vivo. CRANAD-28's imaging brightness, ability to penetrate the blood brain barrier, and low toxicity make the compound a potentially potent imaging tool in Alzheimer's research. In this study, the Aß-labeling ability of CRANAD-28 was investigated in further detail using histological staining to assess different criteria, including stained Aß plaque brightness, Aß plaque size, and Aß plaque number count. The results of this study demonstrated CRANAD-28 to be superior across all criteria assessed. Furthermore, CRANAD-28 and IBA-1 antibody were used to label Aß-plaques and microglia respectively. Statistical analysis with Spearman regression revealed a statistically significant negative correlation between the size of labeled Aß plaques and surrounding microglia density. This finding provides interesting insight into Aß plaque and microglia dynamism in AD pathology and corroborates the findings of previous studies. In addition, we found that CRANAD-28 provided distinct spectral signatures for Aßs in the core and periphery of the plaques. Based on the study's results, CRANAD-28 could be considered as an alternative standard for imaging Aß-plaques in future research studies.


Assuntos
Compostos de Boro/química , Encéfalo/ultraestrutura , Curcumina/química , Corantes Fluorescentes/química , Microglia/ultraestrutura , Placa Amiloide/ultraestrutura , Doença de Alzheimer , Animais , Benzotiazóis/química , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Microscopia Confocal , Microtomia , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Coloração e Rotulagem/métodos
16.
Nat Commun ; 11(1): 8, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911630

RESUMO

Biological tissues exhibit complex spatial heterogeneity that directs the functions of multicellular organisms. Quantifying protein expression is essential for elucidating processes within complex biological assemblies. Imaging mass spectrometry (IMS) is a powerful emerging tool for mapping the spatial distribution of metabolites and lipids across tissue surfaces, but technical challenges have limited the application of IMS to the analysis of proteomes. Methods for probing the spatial distribution of the proteome have generally relied on the use of labels and/or antibodies, which limits multiplexing and requires a priori knowledge of protein targets. Past efforts to make spatially resolved proteome measurements across tissues have had limited spatial resolution and proteome coverage and have relied on manual workflows. Here, we demonstrate an automated approach to imaging that utilizes label-free nanoproteomics to analyze tissue voxels, generating quantitative cell-type-specific images for >2000 proteins with 100-µm spatial resolution across mouse uterine tissue sections preparing for blastocyst implantation.


Assuntos
Automação/métodos , Espectrometria de Massas/métodos , Proteínas/química , Proteômica/métodos , Útero/química , Animais , Feminino , Microdissecção e Captura a Laser , Camundongos , Camundongos Endogâmicos C57BL , Microtomia , Proteínas/genética , Proteínas/metabolismo , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Útero/metabolismo
17.
Am J Respir Cell Mol Biol ; 62(6): 681-691, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31991090

RESUMO

Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.


Assuntos
Pneumopatias/patologia , Pulmão/patologia , Microtomia/métodos , Manejo de Espécimes/métodos , Animais , Modelos Animais de Doenças , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Humanos , Fibrose Pulmonar Idiopática/patologia , Neoplasias Pulmonares/patologia , Camundongos , Doença Pulmonar Obstrutiva Crônica/patologia
18.
Cardiovasc Pathol ; 45: 107177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31891881

RESUMO

BACKGROUND: Coronary artery stenting has become a common procedure and cardiovascular pathology specimens containing these metallic stents are accordingly becoming common. Histologic examination of stented vessels is imperative, but special techniques are needed due to the presence of metal within the tissue. We describe a rapid and inexpensive method for preparing stented vascular specimens for routine histology suitable for use in almost any histology laboratory. DESIGN: After formalin fixation and decalcification, stented vascular segments were freeze-embedded and sectioned using a handheld power micro cutoff wheel tool into ~1 mm slices. Sections were allowed to thaw and the strut shards removed with fine forceps. No longer containing metal, the sections were processed for routine paraffin embedding, microtomy and staining. RESULTS: Histologic sections showed only minor tissue disruption around the stent struts. In our experience with 25 stented arteries (mean interval from implantation 5.6 years), the mean subjective section quality score was 4.1 out of 5. The position of each strut could easily be determined, along with neointimal in-stent restenosis and thrombosis. Local reaction to each strut could be surmised even if minor tissue disruption occurred. The entire process was completed in 2-3 days. The incremental cost over that of routine histology is nominal. CONCLUSION: This method for examining stented vascular segments histologically could readily be applied in most pathology laboratories and serves as a highly practical solution to dilemma of examining stents histologically.


Assuntos
Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/terapia , Reestenose Coronária/patologia , Vasos Coronários/patologia , Metais , Intervenção Coronária Percutânea/instrumentação , Manejo de Espécimes , Stents , Trombose/patologia , Reestenose Coronária/etiologia , Técnica de Descalcificação , Humanos , Microtomia , Inclusão em Parafina , Intervenção Coronária Percutânea/efeitos adversos , Desenho de Prótese , Coloração e Rotulagem , Trombose/etiologia , Fatores de Tempo , Fixação de Tecidos , Fluxo de Trabalho
19.
Biomater Sci ; 8(3): 837-845, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31790090

RESUMO

Semiconductor quantum dots (QDs) have demonstrated utility in long-term single particle tracking of membrane proteins in live cells in culture. To extend the superior optical properties of QDs to more physiologically relevant cell platforms, such as acute brain slices, we examine the photophysics of compact ligand-conjugated CdSe/CdS QDs using both ensemble and single particle analysis in brain tissue media. We find that symmetric core passivation is critical for both photostability in oxygenated media and for prolonged single particle imaging in brain slices. We then demonstrate the utility of these QDs by imaging single dopamine transporters in acute brain slices, achieving 20 nm localization precision at 10 Hz frame rates. These findings detail design requirements needed for new QD probes in complex living environments, and open the door to physiologically relevant studies that capture the utility of QD probes in acute brain slices.


Assuntos
Química Encefálica , Proteínas/química , Pontos Quânticos/química , Animais , Encéfalo/metabolismo , Ligantes , Camundongos , Microscopia de Fluorescência , Microtomia , Proteínas/metabolismo , Compostos de Selênio/química , Coloração e Rotulagem , Compostos de Zinco/química
20.
Med Mol Morphol ; 53(1): 7-14, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31104131

RESUMO

We examined the ultrastructure of the anterior cruciate ligament and assessed age-related changes by comparing the ligaments of young and old monkeys. Ultrathin sections of the anterior cruciate ligament were observed by transmission electron microscopy. The three-dimensional architecture of collagen fibers in the ligament was examined by scanning electron microscopy after tissue specimens were treated with 2 N NaOH to digest the extracellular matrix. At the surface layer of the cruciate ligament in young monkeys, fusiform-shaped fibroblasts actively produced collagen fibrils. The ligament consisted of parallel bundles of dense collagen fibrils of approximately 200 nm in diameter. Collagen fibrils appeared to run linearly. Ligament fibrocytes in the deep layer had a stellate form. Ligament fibrocytes decreased in number and showed marked atrophy in old age. Collagen fibrils had a looser configuration in older monkeys. Despite atrophy of fibroblasts in the deep layer of the anterior cruciate ligament, the area with atrophic fibroblasts in the ligament expands with age, which can likely cause deterioration of and a reduction in collagen fibers. This information can be applied in studies on the cause of the low repair ability of and aging-related changes in the anterior cruciate ligament in humans.


Assuntos
Envelhecimento/fisiologia , Ligamento Cruzado Anterior/ultraestrutura , Colágeno/ultraestrutura , Fibroblastos/ultraestrutura , Articulação do Joelho/ultraestrutura , Animais , Macaca fuscata , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microtomia
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