scholarly journals Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section.

1993 ◽  
Vol 41 (4) ◽  
pp. 489-498 ◽  
Author(s):  
A Trembleau ◽  
D Roche ◽  
A Calas
Keyword(s):  
In Situ Hybridization ◽  
Brain Tissue ◽  
Tissue Section ◽  
Cryostat Section ◽  
Simultaneous Detection ◽  
New Method ◽  
Simple Method ◽  
Brain Tissue Section ◽  
Labeled Probe

We describe here a simple method for combining non-radioactive and radioactive in situ hybridization and immunohistochemistry on the same brain tissue section. This approach was first developed on the well-characterized hypothalamo-neurohypophyseal system, facilitating the optimization of the triple-labeling procedure and the verification of labeling specificity. We report the simultaneous detection of vasopressin (VP) mRNA with a digoxigenin-labeled oligonucleotide, oxytocin (OT) mRNA with a 35S-labeled oligonucleotide, and OT peptide in the same 12-microns cryostat section. This was performed on floating sections as follows: first, the two probes were hybridized simultaneously; second, the peptide was detected with an immunoperoxidase-DAB procedure; third, the digoxigenin-labeled probe was detected with an alkaline phosphatase-NBT/BCIP technique; and finally, the 35S-labeled probe was detected by histological autoradiography. We also demonstrate that this approach is suitable for the simultaneous detection of tyrosine hydroxylase and two less abundant mRNAs, vasoactive intestinal peptide and vasopressin mRNAs, in the suprachiasmatic nucleus. The combination of the three techniques did not significantly diminish their specificity or sensitivity. In conclusion, this new method, permitting the simultaneous detection of three different products of gene expression in the same section, could be useful for further analysis of the phenotypic organization and its plasticity in endocrine or neural tissues.

scholarly journals Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.

1989 ◽  
Vol 108 (6) ◽  
pp. 2343-2353 ◽  
Author(s):  
R H Singer ◽  
G L Langevin ◽  
J B Lawrence
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Colloidal Gold ◽  
Messenger Rna ◽  
Signal To Noise Ratio ◽  
Simultaneous Detection ◽  
Gold Particles ◽  
Rna Molecules ◽  
Nucleic Acid Molecule

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

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

scholarly journals A New Method to Extract Nuclei from Paraffin-Embedded Tissue to Study Lymphomas Using Interphase Fluorescence in Situ Hybridization

2002 ◽  
Vol 160 (6) ◽  
pp. 1967-1972 ◽  
Author(s):  
Sarah F. Paternoster ◽  
Stephanie R. Brockman ◽  
Rebecca F. McClure ◽  
Ellen D. Remstein ◽  
Paul J. Kurtin ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
New Method

scholarly journals Freeze-drying Allows Double Nonradioactive ISH and Antigenic Labeling

2000 ◽  
Vol 48 (4) ◽  
pp. 499-507 ◽  
Author(s):  
Huguette Louis ◽  
Julie Lavie ◽  
Patrick Lacolley ◽  
Danièle Daret ◽  
Jacques Bonnet ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Freeze Drying ◽  
Simultaneous Detection ◽  
Tissue Preservation ◽  
Reliable Technique ◽  
Mrna Detection ◽  
Nonradioactive In Situ Hybridization

Because tissue freeze-drying is an excellent way to preserve antigenic conformation, we have tested the feasibility of this technique to reveal nonradioactive in situ hybridization (ISH) of tissue mRNA. We have compared mRNA detection after different methods of tissue preservation, freeze-drying, cryosectioning, and formaldehyde or methanol fixation. Our results show that nonradioactive ISH is more sensitive for tissues preserved by freeze-drying than for other tissue preparations. We have demonstrated that freeze-drying allows combination of ISH and immunohistochemistry for simultaneous detection of mRNA and antigen because with this technique of tissue preservation ISH does not affect the sensitivity or the amount of the detected antigens. This work underscores the fact that tissue freeze-drying is an easy, convenient, and reliable technique for both ISH and immunohistochemistry and achieves excellent structural conditions for nonradioactive detection.

scholarly journals In Situ Hybridization on Brain Tissue

2003 ◽  
pp. 229-246
Author(s):  
Claas-Hinrich Lammers ◽  
Yoshinobu Hara ◽  
M. Maral Mouradian
Keyword(s):  
In Situ Hybridization ◽  
Brain Tissue

scholarly journals Detection of Canine Parvovirus in Naturally Infected Dogs with Enteritis and Myocarditis by in Situ Hybridization

1997 ◽  
Vol 9 (3) ◽  
pp. 255-260 ◽  
Author(s):  
Whan-Gook Nho ◽  
Jung-Hyang Sur ◽  
Alan R. Doster ◽  
Soon-Bok Kim
Keyword(s):  
In Situ Hybridization ◽  
Capsid Protein ◽  
Dna Sequences ◽  
Middle Part ◽  
Canine Parvovirus ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Liver And Kidney ◽  
Labeled Probe

An improved method for the diagnosis of canine parvovirus using in situ hybridization in standard formalin-fixed, paraffin-embedded tissue sections was developed. A digoxigenin-labeled probe complementary to DNA sequences that code for the entire sequence of the capsid protein VP-1 and the middle part of the sequence of the capsid protein VP-2 was designed. Specific histologic localization of canine parvovirus-infected cells was demonstrated in small intestine, tonsil, lymph node, thymus, spleen, heart, liver, and kidney from dogs diagnosed at necropsy with canine parvovirus infection. The in situ hybridization accurately pinpointed the specific sites of viral infection. The detection of canine parvovirus in liver, kidney, and heart tissues together in the same pups could represent an enhanced virulence of this strain of canine parvovirus and suggests a broadened tissue tropism not seen before in Korean strains of canine parvovirus.

Simultaneous detection of protozoa in the tissues of snakes by double in situ hybridization

2008 ◽  
Vol 71 (4) ◽  
pp. 257-259 ◽  
Author(s):  
Barbara Richter ◽  
Karin Fragner ◽  
Herbert Weissenböck
Keyword(s):  
In Situ Hybridization ◽  
Simultaneous Detection
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