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Journal of Visualized Experiments : JoVE Aug 2020Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are...
Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.
Topics: Animals; Brain; Cryoultramicrotomy; Eosine Yellowish-(YS); Female; Fluorescence; Formaldehyde; Hematoxylin; Immunohistochemistry; Liver; Male; Mice; Polymers; Staining and Labeling
PubMed: 32925894
DOI: 10.3791/61622 -
Nature Communications Dec 2019Single-cell RNA-seq is a powerful tool in decoding the heterogeneity in complex tissues by generating transcriptomic profiles of the individual cell. Here, we report a...
Single-cell RNA-seq is a powerful tool in decoding the heterogeneity in complex tissues by generating transcriptomic profiles of the individual cell. Here, we report a single-nuclei RNA-seq (snRNA-seq) transcriptomic study on human retinal tissue, which is composed of multiple cell types with distinct functions. Six samples from three healthy donors are profiled and high-quality RNA-seq data is obtained for 5873 single nuclei. All major retinal cell types are observed and marker genes for each cell type are identified. The gene expression of the macular and peripheral retina is compared to each other at cell-type level. Furthermore, our dataset shows an improved power for prioritizing genes associated with human retinal diseases compared to both mouse single-cell RNA-seq and human bulk RNA-seq results. In conclusion, we demonstrate that obtaining single cell transcriptomes from human frozen tissues can provide insight missed by either human bulk RNA-seq or animal models.
Topics: Aged; Aged, 80 and over; Cell Nucleus; Female; Frozen Sections; Gene Expression Profiling; Healthy Volunteers; Humans; Male; Photoreceptor Cells; RNA-Seq; Single-Cell Analysis
PubMed: 31848347
DOI: 10.1038/s41467-019-12917-9 -
Canadian Journal of Surgery. Journal... 2023
Topics: Humans; Referral and Consultation; Intraoperative Period; Frozen Sections
PubMed: 36813300
DOI: 10.1503/cjs.001623 -
Archives of Pathology & Laboratory... Jun 2018
Topics: Frozen Sections; Humans; Intraoperative Period; Lymph Nodes; Saline Solution; Specimen Handling
PubMed: 29848030
DOI: 10.5858/arpa.2017-0523-LE -
Fungal Genetics and Biology : FG & B Dec 2009Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been...
Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.
Topics: Biological Transport; Cell Fractionation; Cell Wall; Cryoultramicrotomy; Cryptococcus neoformans; Cytoplasmic Vesicles; Extracellular Space; Immunohistochemistry; Intracellular Membranes; Membrane Lipids; Microscopy, Electron, Transmission; Organelles; Polysaccharides; Virulence Factors
PubMed: 19747978
DOI: 10.1016/j.fgb.2009.09.001 -
Archivio Italiano Di Urologia,... Sep 2021The widespread use of ultrasonography for the investigation of common urological conditions, such as infertility or pain, has resulted in an increased incidence of... (Review)
Review
OBJECTIVES
The widespread use of ultrasonography for the investigation of common urological conditions, such as infertility or pain, has resulted in an increased incidence of incidental non-palpable testicular masses. The majority of these are expected to be benign therefore a conservative approach, either active monitoring or organsparing approach, is recommended. However, there are no clinical or radiological parameters which define the exact nature of such lesions and optimal patient selection criteria are lacking. In this comprehensive review we discuss the significance of incidental, small testicular masses (STMs) and the role of organ-sparing approach in the management of these lesions.
MATERIALS AND METHODS
A non-systematic search was performed using PubMed to identify articles that covered the following topics; clinical implications at diagnosis, role of imaging in identifying the malignant capabilities of a lesion, role of surgery and the final pathology.
RESULTS
Incidental STMs are routinely identified following ultrasound examination of infertile men. STMs usually measure a few millimeters in size and the majority of these are benign. Therefore, strict follow up or an organ-sparing approach, with utilisation of frozen section analysis (FSA), is favored for STMs. FSA has a high correlation with final pathology and prevents unnecessary orchidectomies. Advances in imaging, namely ultrasound and magnetic resonance imaging may provide enhanced assessment of STMs and guidance intraoperatively.
CONCLUSIONS
The optimal approach is not well defined and there is no specific clinical parameter that can predict the nature of STMs. The increasing incidence of small, benign testicular masses has resulted in the development of organ-sparing surgery to investigate and manage these lesions. Organ-sparing surgery has been shown to be practical and carries excellent oncological outcomes.
Topics: Frozen Sections; Humans; Male; Orchiectomy; Patient Selection; Testicular Neoplasms; Ultrasonography
PubMed: 34839628
DOI: 10.4081/aiua.2021.3.296 -
BJU International Nov 2022To investigate the impact of intra-operative neurovascular structure-adjacent frozen-section examination (NeuroSAFE) on the rate of nerve-sparing surgery (NSS) and... (Review)
Review
OBJECTIVES
To investigate the impact of intra-operative neurovascular structure-adjacent frozen-section examination (NeuroSAFE) on the rate of nerve-sparing surgery (NSS) and oncological outcome in a large radical prostatectomy (RP) cohort.
PATIENTS AND METHODS
Between January 2016 and December 2020, 1756 prostate cancer patients underwent robot-assisted RP, of whom 959 (55%) underwent this with NeuroSAFE and 797 (45%) without (control cohort). In cases where NeuroSAFE showed tumour in the margin, a secondary resection was performed. The effect of NeuroSAFE on NSS and positive surgical margin (PSM) status was analysed using logistic regression. Cox regression was used to identify predictors of biochemical recurrence-free survival (BCRFS).
RESULTS AND LIMITATIONS
Patients in the NeuroSAFE cohort had a higher tumour grade (P < 0.001) and clinical stage (P < 0.001) than those in the control cohort. NeuroSAFE enabled more frequent NSS for both pT2 (93% vs 76%; P < 0.001) and pT3 disease (83% vs 55%; P < 0.001). In adjusted analysis, NeuroSAFE resulted in more frequent unilateral (odds ratio [OR] 3.90, 95% confidence interval (CI) 2.90-5.30; P < 0.001) and bilateral (OR 5.22, 95% CI 3.90-6.98; P < 0.001) NSS. While the PSM rate decreased from 51% to 42% in patients with pT3 stage disease (P = 0.031), NeuroSAFE was not an independent predictor of PSM status (OR 0.85, 95% CI 0.68-1.06; P = 0.2) in the entire cohort. Patients who underwent NeuroSAFE had better BCRFS compared to the control cohort (hazard ratio 0.62, 95% CI 0.45-0.84; P = 0.002). This study is limited by its comparison with a historical cohort and lack of functional outcomes.
CONCLUSIONS
NeuroSAFE enables more unilateral and bilateral NSS without negatively affecting surgical margin status and biochemical recurrence. This validation study provides a comprehensive overview of the implementation, evaluation and intra-operative decision making associated with NeuroSAFE in clinical practice.
Topics: Male; Humans; Prostatectomy; Prostate; Frozen Sections; Prostatic Neoplasms; Robotic Surgical Procedures; Margins of Excision
PubMed: 35536200
DOI: 10.1111/bju.15771 -
Methods in Molecular Biology (Clifton,... 2012This chapter describes detailed methods used for laser capture microdissection (LCM) of discrete subpopulations of cells. Topics covered include preparing tissue blocks,...
This chapter describes detailed methods used for laser capture microdissection (LCM) of discrete subpopulations of cells. Topics covered include preparing tissue blocks, cryostat sectioning, processing slides, performing the LCM, and purification of RNA from LCM samples. Notes describe the fine points of each operation, which can often mean the difference between success and failure.
Topics: Animals; Cryoultramicrotomy; Equipment Design; Kidney; Laser Capture Microdissection; RNA
PubMed: 22639264
DOI: 10.1007/978-1-61779-851-1_19 -
Asian Pacific Journal of Cancer... Feb 2019Glioma is the commonest primary intracranial tumour and it has been the most predominant tumour in many studies. It accounts for 24.7% of all primary brain tumour and... (Review)
Review
Glioma is the commonest primary intracranial tumour and it has been the most predominant tumour in many studies. It accounts for 24.7% of all primary brain tumour and 74.6% of malignant brain tumour. Intraoperative diagnosis plays a crucial role in determining the patient management. Frozen section has been the established technique in providing rapid and accurate intraoperative diagnosis. However due to some disadvantages like ice crystal artefact, high expenditure and requirement of skilled technician, there is increase usage of cytology smear either replacing or supplementing frozen section technique. The aim of this review is to determine the diagnostic accuracy of cytology smear and frozen section in glioma and to see whether there is significant difference between those techniques. The overall diagnostic accuracy for frozen section in glioma ranging from 78.4% to 95% while for cytology smear, the diagnostic accuracy ranging from 50% to 100%. Based on certain literatures, no statistically difference was observed in diagnostic accuracy of cytology smear and frozen section. Thus, cytology smear provides an alternative method in establishing intraoperative diagnosis. Both cytology smear and frozen section are complimentary to each other. It is recommended to use both techniques to improve the diagnostic accuracy in addition with adequate knowledge, clinical history, neuroimaging and intraoperative findings.
Topics: Brain Neoplasms; Cytodiagnosis; Frozen Sections; Glioma; Humans; Intraoperative Period
PubMed: 30803189
DOI: 10.31557/APJCP.2019.20.2.321 -
Cold Spring Harbor Protocols Sep 2021The embryo is a classical vertebrate model for molecular, cellular, and developmental biology. Despite many advantages of this organism, such as large egg size and...
The embryo is a classical vertebrate model for molecular, cellular, and developmental biology. Despite many advantages of this organism, such as large egg size and external development, imaging of early embryonic stages is challenging because of nontransparent cytoplasm. Staining and imaging of thin tissue sections is one way to overcome this limitation. Here we describe a step-by-step protocol that combines cryosectioning of gelatin-embedded embryos with immunostaining and imaging. The purpose of this protocol is to examine various cellular and tissue markers after the manipulation of protein function. This protocol can be performed within a 2-d period and allows detection of many antigens by immunofluorescence.
Topics: Animals; Cryoultramicrotomy; Embryo, Mammalian; Embryo, Nonmammalian; Embryonic Development; Fluorescent Antibody Technique; Xenopus laevis
PubMed: 34244349
DOI: 10.1101/pdb.prot107151