In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

1986 ◽  
Vol 18 (11-12) ◽  
pp. 597-604 ◽  
Author(s):  
Heinz Hoefler ◽  
Henry Childers ◽  
Marc R. Montminy ◽  
Ronald M. Lechan ◽  
Richard H. Goodman ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Antisense Rna ◽  
Tissue Sections ◽  
Rna Probes

scholarly journals Localization of cells producing erythropoietin in murine liver by in situ hybridization [see comments]

Blood ◽  
1991 ◽  
Vol 77 (11) ◽  
pp. 2497-2503 ◽  
Author(s):  
ST Koury ◽  
MC Bondurant ◽  
MJ Koury ◽  
GL Semenza
Keyword(s):  
In Situ Hybridization ◽  
Transgenic Mice ◽  
Antisense Rna ◽  
Interstitial Cells ◽  
Cell Type ◽  
Isolated Cells ◽  
Rna Probes ◽  
Sense Strand ◽  
Murine Liver

Abstract In situ hybridization using antisense RNA probes was used to localize cells that produce erythropoietin (EPO) in the livers of anemic transgenic mice expressing the human EPO gene and in livers of anemic nontransgenic mice. In transgenic mice bled from a hematocrit of 55% to one of 10%, hepatocytes surrounding central veins synthesized large amounts of human EPO mRNA. EPO-producing cells were very rare in the area of portal triads. In transgenic mice bled to a hematocrit of 20%, a similar number and distribution of cells contained human EPO mRNA as was found with a 10% hematocrit, but the cells were less heavily labeled, indicating increased EPO production per cell at 10% hematocrit as compared with 20% hematocrit. No human EPO mRNA was detected in the kidneys of anemic transgenic mice, although endogenous murine EPO mRNA was strongly expressed in cortical interstitial cells. In sections of livers from nontransgenic mice bled from a hematocrit of 45% to one of 10%, only isolated cells produced EPO. When the types of cells could clearly be identified, approximately 80% of these cells were hepatocytes, while 20% had a nonepithelial morphology and were located in or adjacent to the sinusoidal spaces. When the sense strand was used as the RNA probe for in situ hybridization, no labeled cells were seen in normal or anemic livers. These results demonstrate that hepatocytes are responsible for production of EPO in both transgenic and nontransgenic mice and that a second cell type that is similar in morphology to EPO-producing interstitial cells in the kidney also produces EPO in the livers of nontransgenic mice.

In Situ Hybridization of Mouse Embryo and Tissue Sections with Radiolabeled RNA Probes

2007 ◽  
Vol 2007 (11) ◽  
pp. pdb.prot4821 ◽  
Author(s):  
Andras Nagy ◽  
Marina Gertsenstein ◽  
Kristina Vintersten ◽  
Richard Behringer
Keyword(s):  
In Situ Hybridization ◽  
Mouse Embryo ◽  
Tissue Sections ◽  
Rna Probes

scholarly journals A fixative suitable for in situ hybridization histochemistry.

1993 ◽  
Vol 41 (6) ◽  
pp. 947-953 ◽  
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.

scholarly journals In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity.

1989 ◽  
Vol 37 (3) ◽  
pp. 389-393 ◽  
Author(s):  
D F Clayton ◽  
A Alvarez-Buylla
Keyword(s):  
In Situ Hybridization ◽  
High Sensitivity ◽  
Cresyl Violet ◽  
High Signal ◽  
Tissue Sections ◽  
Glass Slides ◽  
Cresyl Violet Staining ◽  
Rna Probes ◽  
Hybridization Protocol

We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.

scholarly journals Localization of cells producing erythropoietin in murine liver by in situ hybridization [see comments]

Blood ◽  
1991 ◽  
Vol 77 (11) ◽  
pp. 2497-2503 ◽  
Author(s):  
ST Koury ◽  
MC Bondurant ◽  
MJ Koury ◽  
GL Semenza
Keyword(s):  
In Situ Hybridization ◽  
Transgenic Mice ◽  
Antisense Rna ◽  
Interstitial Cells ◽  
Cell Type ◽  
Isolated Cells ◽  
Rna Probes ◽  
Sense Strand ◽  
Murine Liver

In situ hybridization using antisense RNA probes was used to localize cells that produce erythropoietin (EPO) in the livers of anemic transgenic mice expressing the human EPO gene and in livers of anemic nontransgenic mice. In transgenic mice bled from a hematocrit of 55% to one of 10%, hepatocytes surrounding central veins synthesized large amounts of human EPO mRNA. EPO-producing cells were very rare in the area of portal triads. In transgenic mice bled to a hematocrit of 20%, a similar number and distribution of cells contained human EPO mRNA as was found with a 10% hematocrit, but the cells were less heavily labeled, indicating increased EPO production per cell at 10% hematocrit as compared with 20% hematocrit. No human EPO mRNA was detected in the kidneys of anemic transgenic mice, although endogenous murine EPO mRNA was strongly expressed in cortical interstitial cells. In sections of livers from nontransgenic mice bled from a hematocrit of 45% to one of 10%, only isolated cells produced EPO. When the types of cells could clearly be identified, approximately 80% of these cells were hepatocytes, while 20% had a nonepithelial morphology and were located in or adjacent to the sinusoidal spaces. When the sense strand was used as the RNA probe for in situ hybridization, no labeled cells were seen in normal or anemic livers. These results demonstrate that hepatocytes are responsible for production of EPO in both transgenic and nontransgenic mice and that a second cell type that is similar in morphology to EPO-producing interstitial cells in the kidney also produces EPO in the livers of nontransgenic mice.

Application of in situ hybridization with a novel phenytoin-labeled probe to conventional formalin-fixed, paraffin-embedded tissue sections

1995 ◽  
Vol 52 (3) ◽  
pp. 309-316 ◽  
Author(s):  
Yoshito Eizuru ◽  
Yoichi Minamishima ◽  
Tadashi Matsumoto ◽  
Toshinari Hamakado ◽  
Mikio Mizukoshi ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Labeled Probe ◽  
Formalin Fixed
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