Freeze-drying Allows Double Nonradioactive ISH and Antigenic Labeling

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.

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Rapid Nonradioactive In-Situ Hybridization for mRNA Detection of Calcineurin in Rat Brain Using PCR DNA Probe.

ACTA HISTOCHEMICA ET CYTOCHEMICA ◽

10.1267/ahc.33.115 ◽

2000 ◽

Vol 33 (2) ◽

pp. 115-122 ◽

Cited By ~ 1

Author(s):

Toshifumi Matsui ◽

Nobuteru Usuda ◽

Hiroyuki Arai ◽

Ayami Nakazawa ◽

Sachio Matsushita ◽

...

Keyword(s):

In Situ Hybridization ◽

Rat Brain ◽

Dna Probe ◽

Mrna Detection ◽

Nonradioactive In Situ Hybridization

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mRNA Detection in Cerebral Vessels by Nonradioactive In Situ Hybridization

Blood-Brain Barrier, ◽

10.1385/1-59259-419-0:451 ◽

2003 ◽

pp. 451-464

Author(s):

David Zagzag ◽

Wai Chan

Keyword(s):

In Situ Hybridization ◽

Cerebral Vessels ◽

Mrna Detection ◽

Nonradioactive In Situ Hybridization

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

The Journal of Cell Biology ◽

10.1083/jcb.108.6.2343 ◽

1989 ◽

Vol 108 (6) ◽

pp. 2343-2353 ◽

Cited By ~ 105

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.

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