In situ Hybridization demonstrates the stability of mRNA in post-mortem rat tissues

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) is a approximately 43-kDa secreted protein that has been shown in bioassays to induce endothelial proliferation, angiogenesis, and capillary hyperpermeability. VPF has been suggested to play an important role in the physiology of normal vasculature. To further elucidate the natural functions of VPF in vivo, the expression of VPF in normal tissues was examined using Northern blot analysis and in situ hybridization histochemistry. VPF mRNA is expressed in the brain, kidney, liver, lung, and spleen of the healthy adult rat. On Northern blots, the relative abundance of VPF mRNA observed in these tissues was highest in the lung and lowest in the spleen. As determined by in situ hybridization, the patterns of VPF expression are organ specific. Hybridization of an antisense VPF probe was concentrated in the cerebellar granule cell layer of the brain and in the glomeruli and tubules of the kidney. In the liver and lung, intense hybridization was observed homogeneously throughout both tissues, demonstrating that VPF mRNA is present in virtually every hepatocyte and pulmonary alveolar cell. Hybridization to the spleen was weaker and more diffuse. The widespread expression and organ-specific distribution of VPF mRNA in normal rat tissues supports the suggestion of an extensive role for this factor in the physiology of normal vasculature.

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Glial cell line-derived neurotrophic factor (GDNF) gene expression in the human brain: A post mortem in situ hybridization study with special reference to Parkinson's disease

Journal of Neural Transmission ◽

10.1007/bf01291789 ◽

1996 ◽

Vol 103 (8-9) ◽

pp. 1043-1052 ◽

Cited By ~ 48

Author(s):

S. Hunot ◽

V. Bernard ◽

B. Faucheux ◽

F. Boissi�re ◽

E. Leguern ◽

...

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Human Brain ◽

Special Reference ◽

Cell Line ◽

Neurotrophic Factor ◽

Post Mortem ◽

Hybridization Study ◽

Gdnf Gene

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A simplified and rapid procedure for in situ hybridization on human, flash-frozen, post-mortem brain and its combination with immunohistochemistry

Journal of Neuroscience Methods ◽

10.1016/s0165-0270(96)00086-6 ◽

1996 ◽

Vol 69 (2) ◽

pp. 213-227 ◽

Cited By ~ 16

Author(s):

Ann E. Kingsbury ◽

E. Louise Bray ◽

Oliver J.F. Foster

Keyword(s):

In Situ Hybridization ◽

Post Mortem ◽

Rapid Procedure ◽

Post Mortem Brain

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Evaluation of Cell Proliferation in Rat Tissues with BrdU, PCNA, Ki-67(MIB-5) Immunohistochemistry and In Situ Hybridization for Histone mRNA

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540305101212 ◽

2003 ◽

Vol 51 (12) ◽

pp. 1681-1688 ◽

Cited By ~ 155

Author(s):

Levan Muskhelishvili ◽

John R. Latendresse ◽

Ralph L. Kodell ◽

Eric B. Henderson

Keyword(s):

Cell Proliferation ◽

In Situ Hybridization ◽

Rat Tissues ◽

Histone Mrna ◽

Ki 67

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MO134COVID-19-ASSOCIATED KIDNEY INJURY IS CHARACTERIZED BY ACUTE TUBULAR NECROSIS AND CAPILLARY CONGESTION WITH EVIDENCE FOR SARS-COV-2 IN THE NEPHRON

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab092.0012 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Antoine Bouquegneau ◽

Pauline Erpicum ◽

Stéphanie Grosch ◽

Lionel Habran ◽

Olivier Hougrand ◽

...

Keyword(s):

Electron Microscopy ◽

Acute Kidney Injury ◽

In Situ Hybridization ◽

Acute Tubular Necrosis ◽

Kidney Injury ◽

Post Mortem ◽

Rt Pcr ◽

N Protein ◽

Tubular Necrosis

Abstract Background and Aims Kidney damage has been reported in COVID-19 patients. Despite numerous reports about COVID-19-associated nephropathy, the factual presence of the SARS-CoV-2 in the renal parenchyma remains controversial. Method We consecutively performed 16 immediate (≤3h) post-mortem renal biopsies in patients diagnosed with COVID-19. Kidney samples from 5 patients who died from sepsis and were free from COVID-19 were used as controls. Samples were methodically evaluated by 3 pathologists. Virus detection in the renal parenchyma was performed in all samples by bulk RNA RT-PCR (E and N1/N2 genes), immunostaining (nCoV2019 N-Protein), fluorescent in situ hybridization (nCoV2019-S) and electron microscopy. Results The mean age of our COVID-19 cohort was 68.2±12.8 years, most of whom were males (68.7%). Proteinuria was observed in 53.3% of cases, while acute kidney injury occurred in 60% of cases. Acute tubular necrosis of variable severity was found in all cases, with no tubular or interstitial inflammation. There was no difference in acute tubular necrosis severity between the patients with COVID-19 versus control samples. Congestion in glomerular and peri-tubular capillaries was respectively observed in 56.3 and 87.5% of patients with COVID-19 compared to 20% of controls, with no evidence of thrombi. The nCoV2019 N-Protein was detected in proximal tubules and also at the basolateral pole of scattered cells of the distal tubules in 9/16 cases. In situ hybridization confirmed these findings. RT-PCR of kidney total RNA detected SARS-CoV-2 N gene in one case. Electron microscopy did not show typical viral inclusions. Conclusion Our immediate post-mortem kidney samples from patients with COVID-19 highlight a congestive pattern of acute kidney injury, with no significant glomerular or interstitial inflammation. Immunostaining and in situ hybridization suggest that SARS-CoV-2 is present in various segments of the nephron.

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A fixative suitable for in situ hybridization histochemistry.

Journal of Histochemistry & Cytochemistry ◽

10.1177/41.6.8315285 ◽

1993 ◽

Vol 41 (6) ◽

pp. 947-953 ◽

Cited By ~ 16

Author(s):

F Uehara ◽

N Ohba ◽

Y Nakashima ◽

T Yanagita ◽

M Ozawa ◽

...

Keyword(s):

In Situ Hybridization ◽

Proteinase K ◽

Optimal Time ◽

Hybridization Histochemistry ◽

Tissue Sections ◽

In Situ Hybridization Histochemistry ◽

Rna Probes ◽

Labeled Rna ◽

The Stability

We compared the morphology and stability of hybridization signals between paraffin sections of rat retina fixed with commonly used 4% paraformaldehyde/PBS and those fixed with a fixative containing glutaraldehyde in in situ hybridization histochemistry, using a digoxigenin-labeled RNA probe complementary for beta-galactoside alpha 2,6-sialyltransferase mRNA. Retinal detachment was frequently observed in the sections fixed with 4% paraformaldehyde-PBS, whereas the morphology was satisfactorily preserved in those fixed with either 0.5% glutaraldehyde, 4% paraformaldehyde-PBS, or 2.5% glutaraldehyde-PBS. Without glutaraldehyde, it was difficult to determine the most appropriate length of proteinase K digestion of tissue sections for facilitating probe penetration, since the optimal time for definite hybridization was variable among the retinal cells in heterogeneous layers. By addition of glutaraldehyde to paraformaldehyde or with glutaraldehyde alone, it was easy to establish the appropriate time for the unmasking procedure, since intense mRNA signals were constant throughout the retina by proteinase K digestion for more than 30-40 min. Using a fixative that causes stronger cross-linking (e.g., glutaraldehyde) is recommended to improve not only the morphology but also the stability of hybridization signals in in situ hybridization histochemistry with paraffin embedding and digoxigenin-labeled RNA probes.

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Characterization of telomeres in Hordeum vulgare chromosomes by in situ hybridization. II. Healed broken chromosomes in telotrisomic 4L and acrotrisomic 4L4S lines

Genome ◽

10.1139/g92-149 ◽

1992 ◽

Vol 35 (6) ◽

pp. 975-980 ◽

Cited By ~ 11

Author(s):

Shaoke Wang ◽

Nora L. V. Lapitan ◽

Marion Roder ◽

Takumi Tsuchiya

Keyword(s):

Arabidopsis Thaliana ◽

In Situ Hybridization ◽

Hordeum Vulgare ◽

Telomeric Sequences ◽

The Absolute ◽

The Cross ◽

The Stability ◽

Different Parts

The ends of barley chromosomes hybridize in situ to the telomeric sequences of Arabidopsis thaliana. It was confirmed that the cross-hybridizing sequences in barley are found at the absolute ends of the chromosomes by exonuclease Bal31 digestion. The Bal31 experiments also indicated that telomere-like sequences do not occur in high copies at interstitial sites in barley. To determine whether healing of broken chromosomes occurred in aneuploid lines of barley containing extra chromosomes with breakages in different parts, in situ hybridization with the A. thaliana telomere on telotrisomic 4L and acrotrisomic 4L4S lines was conducted. Telosome 4L possesses breaks in the centromere and in an interstitial location in the long arm, while acrosome 4L4S possesses interstitial breaks in both long and short arms. In situ hybridization revealed the presence of telomere sequences on both broken ends of telosome 4L and acrosome 4L4S. In telosome 4L, telomere sequences were present even at the broken site of the centromere. These results show that broken ends of barley chromosomes were healed. Such healing may explain the stability of these chromosomes through many generations.Key words: telomere, centromere, telosome, acrosome, acrotrisomic, telotrisomic.

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