scholarly journals A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.

1993 ◽  
Vol 41 (1) ◽  
pp. 7-12 ◽  
Author(s):  
J H Wijsman ◽  
R R Jonker ◽  
R Keijzer ◽  
C J van de Velde ◽  
C J Cornelisse ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Staining Method ◽  
Morphological Characteristics ◽  
Image Cytometry ◽  
Apoptotic Cells ◽  
Dna Breaks ◽  
Paraffin Sections ◽  
Tissue Sections

Apoptosis (programmed cell death) can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. We describe a new staining method for formalin-fixed, paraffin-embedded tissue sections that involves an in situ end-labeling (ISEL) procedure. After protease treatment to permeate the tissue sections, biotinylated nucleotides are in situ incorporated into DNA breaks by polymerase and subsequently stained with DAB via peroxidase-conjugated avidin. Staining of cells with the morphological characteristics of apoptosis was demonstrated in tissues known to exhibit programmed cell death, i.e., prostate and uterus after castration, tumors, lymph node follicles, and embryos. Apoptotic cells could be discriminated morphologically from areas of labeled necrotic cells, in which DNA degradation also occurs. Because apoptosis is relatively easily recognized in H&E-stained sections of involuting prostates of castrated rats, we used this model system to validate the ISEL method for the quantification of apoptotic cells. A high correlation was found between the fractions of ISEL-labeled cells and the fractions of apoptotic cells that were morphologically determined in adjacent sections. We conclude that ISEL is a useful technique for quantification of apoptosis in paraffin sections, especially for those tissues in which morphological determination is difficult. Furthermore, this new staining method enables the use of automated image cytometry for evaluating apoptosis.

Assignment1 of the programmed cell death 4 gene (PDCD4) to human chromosome band 10q24 by in situ hybridization

1999 ◽  
Vol 87 (1-2) ◽  
pp. 113-114 ◽  
Author(s):  
H. Soejima ◽  
O. Miyoshi ◽  
H. Yoshinaga ◽  
Z. Masaki ◽  
I. Ozaki ◽  
...  
Keyword(s):  
Cell Death ◽  
In Situ Hybridization ◽  
Programmed Cell Death ◽  
Human Chromosome ◽  
Chromosome Band ◽  
Programmed Cell Death 4 ◽  
Human Chromosome Band

scholarly journals Comedo-Ductal carcinoma in situ: A paradoxical role for programmed cell death

2008 ◽  
Vol 7 (11) ◽  
pp. 1774-1782 ◽  
Author(s):  
Malathy P.V. Shekhar ◽  
Larry Tait ◽  
Robert J. Pauley ◽  
Gen Sheng Wu ◽  
Steven J. Santner ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Ductal Carcinoma In Situ ◽  
Carcinoma In Situ ◽  
Ductal Carcinoma

scholarly journals Ca2+-dependent nuclease is involved in DNA degradation during the formation of the secretory cavity by programmed cell death in fruit of Citrus grandis ‘Tomentosa’

2020 ◽  
Vol 71 (16) ◽  
pp. 4812-4827 ◽  
Author(s):  
Mei Bai ◽  
Minjian Liang ◽  
Bin Huai ◽  
Han Gao ◽  
Panpan Tong ◽  
...  
Keyword(s):  
Cell Death ◽  
In Situ Hybridization ◽  
Programmed Cell Death ◽  
Nuclear Dna ◽  
Expression Patterns ◽  
Citrus Fruit ◽  
Dna Degradation ◽  
Secretory Cavity ◽  
Spatio Temporal

Abstract The secretory cavity is a typical structure in Citrus fruit and is formed by schizolysigeny. Previous reports have indicated that programmed cell death (PCD) is involved in the degradation of secretory cavity cells in the fruit, and that the spatio-temporal location of calcium is closely related to nuclear DNA degradation in this process; however, the molecular mechanisms underlying this Ca2+ regulation remain largely unknown. Here, we identified CgCaN that encodes a Ca2+-dependent DNase in the fruit of Citrus grandis ‘Tomentosa’, the function of which was studied using calcium ion localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The results suggested that the full-length cDNA of CgCaN contains an ORF of 1011 bp that encodes a protein 336 amino acids in length with a SNase-like functional domain. CgCaN digests dsDNA at neutral pH in a Ca2+-dependent manner. In situ hybridization signals of CgCaN were particularly distributed in the secretory cavity cells. Ca2+ and Ca2+-dependent DNases were mainly observed in the condensed chromatin and in the nucleolus. In addition, spatio-temporal expression patterns of CgCaN and its protein coincided with the time-points that corresponded to chromatin degradation and nuclear rupture during the PCD in the development of the fruit secretory cavity. Taken together, our results suggest that Ca2+-dependent DNases play direct roles in nuclear DNA degradation during the PCD of secretory cavity cells during Citrus fruit development. Given the consistency of the expression patterns of genes regulated by calmodulin (CaM) and calcium-dependent protein kinases (CDPK) and the dynamics of calcium accumulation, we speculate that CaM and CDPK proteins might be involved in Ca2+ transport from the extracellular walls through the cytoplasm and into the nucleus to activate CgCaN for DNA degradation.

scholarly journals Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections.

1993 ◽  
Vol 41 (7) ◽  
pp. 1023-1030 ◽  
Author(s):  
R Gold ◽  
M Schmied ◽  
G Rothe ◽  
H Zischler ◽  
H Breitschopf ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Specific Cell ◽  
Nick Translation ◽  
Proliferating Cells ◽  
Cell Surface Antigens ◽  
Simultaneous Application ◽  
Tissue Sections ◽  
In Situ Nick Translation

Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level. We first established the technique for cell preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin. The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT. Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT. Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes. Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis. However, this technique was not useful in detecting early stages of necrotic cell death.

Genetic Loci Controlling Lethal Cell Death in Tomato Caused by Viral Satellite RNA Infection

2012 ◽  
Vol 25 (8) ◽  
pp. 1034-1044 ◽  
Author(s):  
Ping Xu ◽  
Hua Wang ◽  
Frank Coker ◽  
Jun-ying Ma ◽  
Yuhong Tang ◽  
...  
Keyword(s):  
Cell Death ◽  
In Situ Hybridization ◽  
Programmed Cell Death ◽  
Minus Strand ◽  
Satellite Rna ◽  
Dominant Trait ◽  
Systemic Necrosis ◽  
Backcross Population ◽  
Major Qtl

Cucumber mosaic virus (CMV) associated with D satellite RNA (satRNA) causes lethal systemic necrosis (LSN) in tomato (Solanum lycopersicum), which involves programmed cell death. No resistance to this disease has been found in tomato. We obtained a line of wild tomato, S. habrochaitis, with a homogeneous non-lethal response (NLR) to the infection. This line of S. habrochaitis was crossed with tomato to generate F1 plants that survived the infection with NLR, indicating that NLR is a dominant trait. The NLR trait was successfully passed on to the next generation. The phenotype and genotype segregation was analyzed in the first backcross population. The analyses indicate that the NLR trait is determined by quantitative trait loci (QTL). Major QTL associated with the NLR trait were mapped to chromosomes 5 and 12. Results from Northern blot and in situ hybridization analyses revealed that the F1 and S. habrochaitis plants accumulated minus-strand satRNA more slowly than tomato, and fewer vascular cells were infected. In addition, D satRNA-induced LSN in tomato is correlated with higher accumulation of the minus-strand satRNA compared with the accumulation of the minus strand of a non-necrogenic mutant D satRNA.

scholarly journals Ultrasound imaging of apoptosis: high-resolution non-invasive monitoring of programmed cell death in vitro, in situ and in vivo

1999 ◽  
Vol 81 (3) ◽  
pp. 520-527 ◽  
Author(s):  
G J Czarnota ◽  
M C Kolios ◽  
J Abraham ◽  
M Portnoy ◽  
F P Ottensmeyer ◽  
...  
Keyword(s):  
Cell Death ◽  
High Resolution ◽  
Programmed Cell Death ◽  
Ultrasound Imaging ◽  
Invasive Monitoring ◽  
Non Invasive

scholarly journals Apoptosis and Necrosis after Reversible Focal Ischemia: An in Situ DNA Fragmentation Analysis

1996 ◽  
Vol 16 (2) ◽  
pp. 186-194 ◽  
Author(s):  
C. Charriaut-Marlangue ◽  
I. Margaill ◽  
A. Represa ◽  
T. Popovici ◽  
M. Plotkine ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Cortical Neurons ◽  
Apoptotic Cell ◽  
Chromatin Condensation ◽  
Focal Ischemia ◽  
Dna Breaks ◽  
Ischemic Lesion ◽  
In Cells

Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain ( Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385–388). We now show that a significant number of striatal and cortical neurons exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.

Apoptosis after traumatic human spinal cord injury

1998 ◽  
Vol 5 (4) ◽  
pp. E1 ◽  
Author(s):  
Evelyne Emery ◽  
Philipp Aldana ◽  
Mary Bartlett Bunge ◽  
William Puckett ◽  
Anu Srinivasan ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Positive Staining ◽  
Apoptotic Cells ◽  
Nuclear Staining ◽  
Human Spinal Cord ◽  
Cord Injury

Object Apoptosis is a form of programmed cell death seen in a variety of developmental and disease states, including traumatic injuries. The main objective of this study was to determine whether apoptosis is observed after human spinal cord injury (SCI). The spatial and temporal expression of apoptotic cells as well as the nature of the cells involved in programmed cell death were also investigated. Methods The authors examined the spinal cords of 15 patients who died between 3 hours and 2 months after a traumatic SCI. Apoptotic cells were found at the edges of the lesion epicenter and in the adjacent white matter, particularly in the ascending tracts, by using histological (cresyl violet, hematoxylin and eosin) and nuclear staining (Hoechst 33342). The suspected presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the interleukin-1-beta-converting enzyme/Caenorhabditis elegans D 3 family of proteases that plays an essential role in programmed cell death. Apoptosis in this series of human SCIs was a prominent pathological finding in 14 of the 15 spinal cords examined when compared with five uninjured control spinal cords. To determine the type of cells undergoing apoptosis, the authors immunostained specimens with a variety of antibodies, including glial fibrillary acidic protein, 2,′3′-cyclic nucleotide 3′-phosphohydrolase (CNPase), and CD45/68. Oligodendrocytes stained with CNPase and a number of apoptotic nuclei colocalized with positive staining for this antibody. Conclusions These results support the hypothesis that apoptosis occurs in human SCIs and is accompanied by the activation of CPP-32 of the cysteine protease family. This mechanism of cell death contributes to the secondary injury processes seen after human SCI and may have important clinical implications for the further development of protease inhibitors to prevent programmed cell death.

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