scholarly journals Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons

2021 ◽  
pp. 002215542110433
Author(s):  
Takuya Ikenari ◽  
Tatsuya Kawaguchi ◽  
Rei Ota ◽  
Miki Matsui ◽  
Ryota Yoshida ◽  
...  
Keyword(s):  
Cell Death ◽  
Glial Cells ◽  
Cell Types ◽  
Staining Procedure ◽  
Double Staining ◽  
Acute Encephalopathy ◽  
Experimental Conditions ◽  
Tissue Sections ◽  
Staining Protocol ◽  
Mature Neurons

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin–biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions:

scholarly journals SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

2020 ◽  
Author(s):  
Alexandros Sountoulidis ◽  
Andreas Liontos ◽  
Hong Phuong Nguyen ◽  
Alexandra B. Firsova ◽  
Athanasios Fysikopoulos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rolling Circle Amplification ◽  
Marker Gene ◽  
Cell Types ◽  
Experimental Conditions ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Tissue Sections ◽  
Specificity And Sensitivity

AbstractChanges in cell identities and positions underlie tissue development and disease progression. Although, single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell-states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single Cell Resolution INSitu Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRISHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity and quantitative qualities of SCRINSHOT facilitate single cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

scholarly journals SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000675
Author(s):  
Alexandros Sountoulidis ◽  
Andreas Liontos ◽  
Hong Phuong Nguyen ◽  
Alexandra B. Firsova ◽  
Athanasios Fysikopoulos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rolling Circle Amplification ◽  
Marker Gene ◽  
Cell Types ◽  
Experimental Conditions ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Tissue Sections ◽  
Specificity And Sensitivity

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

Ultrasturcture of cerebellar cells and neuropil in rats subjected to partial global cerebral ischemia

1986 ◽  
Vol 44 ◽  
pp. 126-127
Author(s):  
R.V.W. Dimlich ◽  
M.H. Biros
Keyword(s):  
Purkinje Cells ◽  
Glial Cells ◽  
Granule Cells ◽  
Molecular Layer ◽  
Cell Types ◽  
Primary Objective ◽  
Global Cerebral Ischemia ◽  
Forebrain Ischemia ◽  
Substantial Evidence ◽  
Cerebellar Cells

Although a previous study in this laboratory determined that Purkinje cells of the rat cerebellum did not appear to be damaged following 30 min of forebrain ischemia followed by 30 min of reperfusion, it was suggested that an increase in rough endoplasmic reticulum (RER) and/or polysomes had occurred in these cells. The primary objective of the present study was to morphometrically determine whether or not this increase had occurred. In addition, since there is substantial evidence that glial cells may be affected by ischemia earlier than other cell types, glial cells also were examined. To ascertain possible effects on other cerebellar components, granule cells and neuropil near Purkinje cells as well as neuropil in the molecular layer also were evaluated in this investigation.

A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

1990 ◽  
Vol 48 (3) ◽  
pp. 378-379
Author(s):  
Jane E. Ramberg ◽  
Shigeto Tohma ◽  
Peter E. Lipsky
Keyword(s):  
B Cells ◽  
Adhesion Molecule ◽  
Colloidal Gold ◽  
Intercellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Staining Procedure ◽  
Double Staining ◽  
T And B Cells ◽  
Microtiter Plates ◽  
Streptavidin Peroxidase

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

scholarly journals The Microbiota–Gut–Brain Axis and Alzheimer Disease. From Dysbiosis to Neurodegeneration: Focus on the Central Nervous System Glial Cells

2021 ◽  
Vol 10 (11) ◽  
pp. 2358
Author(s):  
Maria Grazia Giovannini ◽  
Daniele Lana ◽  
Chiara Traini ◽  
Maria Giuliana Vannucchi
Keyword(s):  
Alzheimer Disease ◽  
Glial Cells ◽  
Cell Types ◽  
The Past ◽  
The Central Nervous System ◽  
Diseased State ◽  
The Brain ◽  
Alzheimer Disease Pathogenesis ◽  
Brain Homeostasis

The microbiota–gut system can be thought of as a single unit that interacts with the brain via the “two-way” microbiota–gut–brain axis. Through this axis, a constant interplay mediated by the several products originating from the microbiota guarantees the physiological development and shaping of the gut and the brain. In the present review will be described the modalities through which the microbiota and gut control each other, and the main microbiota products conditioning both local and brain homeostasis. Much evidence has accumulated over the past decade in favor of a significant association between dysbiosis, neuroinflammation and neurodegeneration. Presently, the pathogenetic mechanisms triggered by molecules produced by the altered microbiota, also responsible for the onset and evolution of Alzheimer disease, will be described. Our attention will be focused on the role of astrocytes and microglia. Numerous studies have progressively demonstrated how these glial cells are important to ensure an adequate environment for neuronal activity in healthy conditions. Furthermore, it is becoming evident how both cell types can mediate the onset of neuroinflammation and lead to neurodegeneration when subjected to pathological stimuli. Based on this information, the role of the major microbiota products in shifting the activation profiles of astrocytes and microglia from a healthy to a diseased state will be discussed, focusing on Alzheimer disease pathogenesis.

scholarly journals TUNEL Assay: A Powerful Tool for Kidney Injury Evaluation

2021 ◽  
Vol 22 (1) ◽  
pp. 412
Author(s):  
Christopher L. Moore ◽  
Alena V. Savenka ◽  
Alexei G. Basnakian
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Cell Injury ◽  
Kidney Injury ◽  
Cell Types ◽  
Tunel Assay ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Hypoxic Injury ◽  
Additional Information ◽  
Tissue Compartments

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay is a long-established assay used to detect cell death-associated DNA fragmentation (3’-OH DNA termini) by endonucleases. Because these enzymes are particularly active in the kidney, TUNEL is widely used to identify and quantify DNA fragmentation and cell death in cultured kidney cells and animal and human kidneys resulting from toxic or hypoxic injury. The early characterization of TUNEL as an apoptotic assay has led to numerous misinterpretations of the mechanisms of kidney cell injury. Nevertheless, TUNEL is becoming increasingly popular for kidney injury assessment because it can be used universally in cultured and tissue cells and for all mechanisms of cell death. Furthermore, it is sensitive, accurate, quantitative, easily linked to particular cells or tissue compartments, and can be combined with immunohistochemistry to allow reliable identification of cell types or likely mechanisms of cell death. Traditionally, TUNEL analysis has been limited to the presence or absence of a TUNEL signal. However, additional information on the mechanism of cell death can be obtained from the analysis of TUNEL patterns.

scholarly journals Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia

2021 ◽  
Vol 22 (9) ◽  
pp. 4334
Author(s):  
Katrina Albert ◽  
Jonna Niskanen ◽  
Sara Kälvälä ◽  
Šárka Lehtonen
Keyword(s):  
Stem Cells ◽  
Neurodegenerative Diseases ◽  
Neurodegenerative Disease ◽  
Induced Pluripotent Stem Cells ◽  
Glial Cells ◽  
Pluripotent Stem Cells ◽  
Cell Types ◽  
Human Ipscs ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

scholarly journals A practical solution to pseudoreplication bias in single-cell studies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kip D. Zimmerman ◽  
Mark A. Espeland ◽  
Carl D. Langefeld
Keyword(s):  
Single Cell ◽  
Mixed Models ◽  
Random Effect ◽  
Cell Types ◽  
Error Rates ◽  
Specific Cell ◽  
Experimental Conditions ◽  
Type 1 Error ◽  
Sample Correlation ◽  
Inflated Type

AbstractCells from the same individual share common genetic and environmental backgrounds and are not statistically independent; therefore, they are subsamples or pseudoreplicates. Thus, single-cell data have a hierarchical structure that many current single-cell methods do not address, leading to biased inference, highly inflated type 1 error rates, and reduced robustness and reproducibility. This includes methods that use a batch effect correction for individual as a means of accounting for within-sample correlation. Here, we document this dependence across a range of cell types and show that pseudo-bulk aggregation methods are conservative and underpowered relative to mixed models. To compute differential expression within a specific cell type across treatment groups, we propose applying generalized linear mixed models with a random effect for individual, to properly account for both zero inflation and the correlation structure among measures from cells within an individual. Finally, we provide power estimates across a range of experimental conditions to assist researchers in designing appropriately powered studies.

scholarly journals Ferroptotic cell death triggered by conjugated linolenic acids is mediated by ACSL1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alexander Beatty ◽  
Tanu Singh ◽  
Yulia Y. Tyurina ◽  
Vladimir A. Tyurin ◽  
Svetlana Samovich ◽  
...  
Keyword(s):  
Cell Death ◽  
Therapeutic Potential ◽  
Neutral Lipids ◽  
Cell Types ◽  
Lipid Hydroperoxides ◽  
Conjugated Linolenic Acids ◽  
Cellular Lipids ◽  
Transcriptional Changes ◽  
Acyl Chains ◽  
Tumor Growth And Metastasis

AbstractFerroptosis is associated with lipid hydroperoxides generated by the oxidation of polyunsaturated acyl chains. Lipid hydroperoxides are reduced by glutathione peroxidase 4 (GPX4) and GPX4 inhibitors induce ferroptosis. However, the therapeutic potential of triggering ferroptosis in cancer cells with polyunsaturated fatty acids is unknown. Here, we identify conjugated linoleates including α-eleostearic acid (αESA) as ferroptosis inducers. αESA does not alter GPX4 activity but is incorporated into cellular lipids and promotes lipid peroxidation and cell death in diverse cancer cell types. αESA-triggered death is mediated by acyl-CoA synthetase long-chain isoform 1, which promotes αESA incorporation into neutral lipids including triacylglycerols. Interfering with triacylglycerol biosynthesis suppresses ferroptosis triggered by αESA but not by GPX4 inhibition. Oral administration of tung oil, naturally rich in αESA, to mice limits tumor growth and metastasis with transcriptional changes consistent with ferroptosis. Overall, these findings illuminate a potential approach to ferroptosis, complementary to GPX4 inhibition.

scholarly journals Double-staining procedure for the fluorescent treponemal antibody absorption (FTA-ABS) test.

1979 ◽  
Vol 55 (2) ◽  
pp. 105-108
Author(s):  
E F Hunter ◽  
R M McKinney ◽  
S E Maddison ◽  
D D Cruce
Keyword(s):  
Staining Procedure ◽  
Double Staining ◽  
Antibody Absorption
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