key: cord-0944225-hsspggp8
authors: Sirinarumitr, Theerapol; Paul, Prem S.; Halbur, Patrick G.; Kluge, John P.
title: Rapid in situ hybridization technique for the detection of ribonucleic acids in tissues using radiolabelled and fluorescein-labelled riboprobes
date: 1997-08-31
journal: Molecular and Cellular Probes
DOI: 10.1006/mcpr.1997.0114
sha: 21d19c09d38d83036d23288d2f7c0ea8dc2ede13
doc_id: 944225
cord_uid: hsspggp8

Abstract In situhybridization (ISH) is a useful diagnostic and research tool, but is also time consuming. This study was conducted to determine if a rate enhancement hybridization (REH) buffer, developed for membrane hybridization, could be used to decrease hybridization time for ISH. Tissue from swine with an enteric disease produced by a swine coronavirus, transmissible gastroenteritis virus (TGEV), was used as a model to standardize hybridization conditions for a rapid ISH technique. Small intestinal sections from pigs experimentally and naturally infected with TGEV were hybridized for various times at 52°C and 70°C with a radiolabelled or a fluorescein-labelled RNA probe in a standard hybridization or a REH buffer. Viral RNA was detected in intestines from as early as 30 min of hybridization by using both buffers with the radiolabelled probe; however, the signal was stronger with the REH buffer. With the fluorescein-labelled probe, viral RNA was detected in virus-infected cells of the intestines after 30 min of hybridization by using the REH buffer. Signal intensity was greater with the REH buffer than with the standard hybridization buffer when compared at each hybridization time and hybridization temperature using both radiolabelled and fluorescein-labelled probes. With the REH buffer, hybridization signal intensity was greater at 70°C than at 52°C for both probes. The best results were obtained when small intestinal sections were hybridized at 70°C for 2 h using a radiolabelled or a fluorescein-labelled probe diluted in the REH buffer. The fluorescein-labelled RNA probe with REH buffer resulted in a minimal non-specific signal when compared with the radiolabelled probe. These studies demonstrated that the REH buffer can be used to decrease the time of ISH for the detection of viral RNA. This rapid ISH technique should have broad applications in the utilization of probe technology in diagnostics and research for the detection of target ribonucleic acidsin situ

(Received 13 November 1996, Accepted 25 April 1997)

In situ hybridization (ISH) is a useful diagnostic and research tool, but is also time consuming. This study was conducted to determine if a rate enhancement hybridization (REH) buffer, developed for membrane hybridization, could be used to decrease hybridization time for ISH. Tissue from swine with an enteric disease produced by a swine coronavirus, transmissible gastroenteritis virus (TGEV), was used as a model to standardize hybridization conditions for a rapid ISH technique. Small intestinal sections from pigs experimentally and naturally infected with TGEV were hybridized for various times at 52°C and 70°C with a radiolabelled or a fluorescein-labelled RNA probe in a standard hybridization or a REH buffer. Viral RNA was detected in intestines from as early as 30 min of hybridization by using both buffers with the radiolabelled probe; however, the signal was stronger with the REH buffer. With the fluorescein-labelled probe, viral RNA was detected in virus-infected cells of the intestines after 30 min of hybridization by using the REH buffer. Signal intensity was greater with the REH buffer than with the standard hybridization buffer when compared at each hybridization time and hybridization temperature using both radiolabelled and fluorescein-labelled probes. With the REH buffer, hybridization signal intensity was greater at 70°C than at 52°C for both probes. The best results were obtained when small intestinal sections were hybridized at 70°C for 2 h using a radiolabelled or a fluorescein-labelled probe diluted in the REH buffer. The fluorescein-labelled RNA probe with REH buffer resulted in a minimal non-specific signal when compared with the radiolabelled probe. These studies demonstrated that the REH buffer can be used to decrease the time of ISH for the detection of viral RNA. This rapid ISH technique should have broad applications in the utilization of probe technology in diagnostics and research for the detection of target ribonucleic acids in situ.© 1997 Academic Press Limited

In situ hybridization (ISH) is widely used for the or oligonucleotides. A major disadvantage of ISH is that it is a time-consuming technique, taking about 2 detection of DNA and RNA in intact eukaryotic and procaryotic cells by using radiolabelled or non-radio-days for non-isotopic ISH and 4 days or more for isotopic ISH. One of the rate limiting steps is hy-labelled probes. Probes for ISH may be DNA, RNA bridization which requires at least 16 h. 1-6 There have ends of the jejunum and the ileum were collected and fixed in 10% neutral buffered formalin. A segment been two reports describing a reduction of hybridization time to 3 h using DNA probes 7 and to 2 h of the small intestine from the five pigs naturally infected with TGEV and the two TGEV negative pigs using ribotypes. 8 Recently, rate enhancement hybridization (REH) buffers have become commercially was placed in 10% neutral buffered formalin. The tissues were processed and embedded in paraffin. available. These buffers can decrease hybridization time for membrane-based procedures such as South-Consecutive sections were cut from each tissue, placed on nuclease-free silylated slides (PGC sci-ern, northern, and dot blot hybridizations.

For the development of rapid ISH, tissues from entific), deparaffinized and hydrated as previously reported. 18 swine with experimentally induced, transmissible gastroenteritis (TGE), were used as a model. TGE is characterized by vomiting, severe diarrhoea, and high mortality in piglets during the first few weeks of life.

Preparation of probes The causative agent of TGE is a coronavirus referred to as transmissible gastroenteritis virus (TGEV). Co-RNA probes were prepared from plasmid pPSP.FP 1 containing the left half of the S gene of TGEV cloned ronavirus particles are pleomorphic and enveloped and contain a single-stranded positive-sense RNA in a phagemid vector. 19 The plasmid pPSP.FP 1 contains nucleotides 1678 to 2250 20 of the S gene of TGEV, genome. 9 TGEV replicates in the cytoplasm of the mature absorptive enterocytes 9-14 and causes villous and was linearized with the restriction enzyme BamHI (Promega). The in vitro transcription and labelling of atrophy. Here we describe a rapid ISH technique, using REH buffer, that allows decreased hybridization the probe was performed as described previously 18 by using T7 RNA polymerase, the four NTPs (ATP, time for both radiolabelled and fluorescein-labelled probes.

CTP labelled RNA probe was labelled by using a RNA colour kit (Amersham) and purified by using a Se-erence virus. [15] [16] [17] phadex G-50 spin column (Boehringer Mannheim). The specificity of probes was determined by dot blot hybridization as previously reported. 21 Source of tissues Four 8.5 week-old TGEV-negative pigs were used. Two pigs were inoculated orally with 10 5 plaque In situ hybridization forming units (pfu) of virulent Miller strain of TGEV and two pigs served as non-inoculated controls. Tis-Deparaffinized tissues were treated and hybridized as reported previously. 18 Briefly, tissues were treated sues from seven pigs submitted as separate diagnostic cases to the Veterinary Diagnostic Laboratory, College with proteinase K (1 g ml −1 , Ameresco) for 15 min at 37°C and acetylated with 0.25% acetic anhydride of Veterinary Medicine, Iowa State University were used to represent TGEV naturally-infected and non-in 0.1  triethanolamine (pH 8.0) for 10 min at room temperature. Sections hybridized with a fluorescein-infected pigs. Five samples were positive and two were negative for TGEV antigen by direct immuno-labelled RNA probe were incubated with 20% (v/v) cold acetic acid for 15 s after treatment with pro-fluorescence of frozen intestinal sections.

teinase K to destroy endogenous alkaline phosphatase. Sections were hybidized with 50 l of standard hybridization buffer (50% formamide, 10% Processing of tissues for in situ hybridization dextran sulfate, 3×SSC, 50 m sodium phosphate, pH 7.4, 1×Denhardt's solution, 0.1 mg ml −1 yeast TGEV-inoculated and non-inoculated pigs were euthanized and necropsied at 3 days post-inoculation.

tRNA, and 10 m DTT) or REH buffer (Rapid-Hyb buffer, Amersham) containing 0.5 l of a 35 S-labelled At the time of necropsy, 1 cm segments of the posterior end of the duodenum, and the anterior and posterior RNA probe (1.43 ng l −1 or 1.6×10 6 cpm l −1 ) or 1 l of a fluorescein-labelled RNA probe (1 ng l −1 ). Controls included: tissue sections from two noninoculated control pigs and two TGEV-free pigs sub-Sections hybridized with a 35 S-labelled RNA probe were divided into 4 groups, which were incubated mitted to the Veterinary Diagnostic Laboratory; RNase-treated tissue sections from TGEV-inoculated with the standard hybridization buffer at 52°C and 70°C, and the REH buffer at 52°C and 70°C. Sections pigs and TGEV-naturally infected pigs; sections from TGEV-infected pigs hybridized with positive strand hybridized with a flourescein-labelled RNA probe were divided into two groups which were hybridized RNA probes; sections from TGEV-infected pigs hybridized with the excess amount of non-labelled RNA with the standard hybridization buffer at 52°C or the REH buffer at 70°C. Each group of sections were probe followed by hybridization with labelled RNA probes. For the fluorescein-labelled RNA probe, we incubated with hybridization buffer for 20 h, 2 h, 1 h or 30 min. Sections were treated with 20 g ml −1 used a hybridization buffer-free probe as an additional control. RNase A (Worthington) at 37°C for 30 min, and washed in 2×SSC for 5 min at 52°C, 1×SSC for 5 min at 52°C, 0.5×SSC for 1 h at 52°C, and 0.5×SSC for 5 min at room temperature.

Autoradiography results were captured on X-ray films Detection of the in situ hybridization signal by an AGFA Arcus II flatbed scanner and edited by using software Adobe Photoshop 3.0 on a McIntosh computer. The results were printed with a video For the 35 S-labelled RNA probe, sections were dehydrated and air dried. X-ray film (BIOMAX MR, printer (Sony Color Video Printer Up-5000). The intensity of the signal was analysed using software NIH Eastman Kodak Co.) was exposed to hybridization sections at −70°C for 24 h, developed and evaluated.

Image 1.58 and reported as mean density and total signal area. For non-isotopic ISH, slides were scored Sections were then coated with Kodak NTB-2 emulsion gel (Eastman Kodak Co.) which was diluted 1:1 by using semiquantitation as follows: −=negative, +=weak, ++=moderate, +++=strong hy-with 600 m ammonium acetate 1,2 and preheated to 42°C. 1-4 These sections were kept in a light-proof box bridization signal. at 4°C for 2 days. The slides were developed at 15°C for 3 min by using developer (Kodak D-19) diluted 1:1 with deionized water, followed by a 20 s rinse in RESULTS deionized water and then fixed for 3 min in fixer (Eastman Kodak Co). Sections were then rinsed with

In situ hybridization of sections of TGEVdeionized water for 5 min 4 and counterstained with infected tissues with 35 S-labelled RNA probe haematoxylin and eosin.

For the fluorescein-labelled RNA probe, sections Preliminary experiment on comparison of the were washed in Tris-buffered saline (TBS) containing effect of two hybridization buffers 100 m Tris-HCl, pH 7.5, and 400 m NaCl for 5 min at room temperature. Sections were incubated for 1 h Studies were first performed by using a radiolabelled at room temperature in a moist chamber with a riboprobe to compare the hybridization using a standblocking solution consisting of 0.5% (w/v) blocking ard hybridization buffer with that using REH buffer. agent (Amersham) in TBS. Sections were then rinsed Hybridization using the standard hybridization buffer with TBS for 1 min and incubated with anti-fluorwas performed at 52°C whereas hybridization with escein antibody conjugated with alkaline phosphatase the REH buffer was performed at 70°C, both for 0.5, (Amersham) diluted 1:400 in 0.5% (w/v) BSA fraction 1 and 2 h. Hybridization signals were detected with V in TBS for 1 h in a moist chamber at room temboth hybridization buffers at all times examined; perature. Sections were washed in TBS three times however, the signal was consistently more intense for 5 min each, and in detection buffer (100 m Triswith the REH buffer than with the standard hy-HCl, pH 9.5, 100 m NaCl, 50 m MgCl 2 ) for 5 min bridization buffer. Intensity of the signal after 2 h of at room temperature. Sections were incubated with hybridization with the REH buffer was greater than substrate (45 l of 4-nitroblue tetrazolium chloride that at 20 h using the standard hybridization buffer. and 35 l of 5-bromo-4-chloro-3-indolyl-phosphate This experiment suggested that the hybridization bufin 10 ml detection buffer) for 1 h in the dark, then rinsed in deionized water and counterstained with fer and temperature may impact hybridization kinetics. nuclear fast red for 2 min. 

REH buffer at 52°C for 20 h (Figs 1 & 2) . The intensity of the hybridization signal increased with time as the two buffers hybridization signal with the REH buffer at 70°C was less intense at 2 h compared to 20 h. However, the To further determine the effect of the temperature and background was also higher after 20 h hybridization the buffers, hybridization was performed using the than after 2 h hybridization. Viral RNA could be standard hybridization and REH buffer each at two detected with the REH buffer at 70°C as early as different temperatures of 52°C and 70°C for 0.5, 1, 30 min. 2 and 20 h of hybridization. Hybridization with the standard hybridization buffer was best at 52°C, whereas for the REH buffer, the optimal hybridization temperature giving the best signal was 70°C (Fig. 1a Microscopic examination of intestinal sections & b). The hybridization signal at 70°C with the REH hybridized with 35 S-labelled RNA probe buffer after 2 h hybridization was equivalent to or better than that detected after 20 h of hybridization at Sections obtained from infected pigs and coated with 52°C with the standard hybridization buffer. Sections emulsion gel had heavy concentrations of silver grains hybridized with the REH buffer for 2 h at 70°C also along the mucosal layer. In contrast, sections from gave the best signal-to-noise ratio and the most intense uninoculated control animals were negative for silver signal when compared with the standard hygrains. The silver grains were primarily concentrated over the cytoplasm of the villous enterocytes (Fig. 3) , bridization buffer at both 52°C and 70°C, and the . Sections in panels A and B were hybridized using standard hybridization buffer at 52°C and those in panel C were hybridized using standard hybridization buffer at 70°C. Sections in panels D and F were hybridized using the REH buffer at 70°C and those in panel E were hybridized using REH buffer at 52°C. The results showed the hybridization signal as dark continuous lines along the mucosal surface of the TGEV-inoculated small intestinal sections. Such a hybridization signal was not detected in small intestines from uninoculated pigs. Best results were obtained with REH buffer after 2 h hybridization at 70°C. and some were present in crypt epithelial cells. Signal nucleus. Sections hybridized with the REH buffer at 70°C clearly had a higher signal intensity and a greater intensity was similar to the autoradiographic results captured on X-ray films. Sections that were hybridized number of viral infected cells than sections hybridized with standard hybridization buffer at 52°C, at each with the REH buffer at 70°C for 2 h had higher concentration of silver grains than those hybridized hybridization time (Table 1 ). Viral infected cells were detected after 2 h of hybridization using standard with the standard hybridization buffer at 52°C for 20 h (Fig. 3a and b) . Sections that hybridized with hybridization buffer at 52°C. In contrast, viral infected cells were detected after 30 min of hybridization the REH buffer had higher non-specific silver grains than standard hybridization buffer, which increased with the REH buffer at 70°C. This treatment gave an equivalent or more intense signal, and a greater with time. The histopathologic changes observed in the small intestines from the infected animals were number of viral infected cells than secretions hybridized with standard hybridization buffer at 52°C villous atrophy, blunted and denuded villi, and replacement of the columnar epithelial cells by flat to for 2 h. Sections hybridized with the REH buffer at 70°C for 20 h had the highest signal intensity, and cuboidal cells.

the greatest number of viral infected cells. There was only a slight increase in signal intensity after 20 h hybridization with REH buffer at 70°C without an In situ hybridization on sections of TGEVinfected tissues using a fluorescein-labelled increase in the number of virus infected cells over those hybridized for 2 h. The signal intensity and the RNA probe number of virus infected cells on sections hybridized with the REH buffer at 70°C for 2 h was equal to or Once the hybridization conditions were standardized with the radiolabelled probe, we compared the effect higher than that with sections hybridized with standard hybridization buffer at 52°C for 20 h (Fig. 4 a & of standard hybridization buffer at 52°C with the REH buffer at 70°C by using a fluorescein-labelled RNA b). The signal intensity and the number of viral nucleic acid positive cells were less after 1 h and 30 min probe. Viral nucleic acid was detected in enterocytes of the small intestinal sections hybridized with either compared to 2 h hybridization. The intensity of the signals varied from purple after 30 min, to dark purple buffer, mainly in villous epithelial cells (Fig. 4) and some crypt epithelial cells. Dark purple colour was after 1 h and to very dark purple after both 2 h and 20 h hybridization. Sections hybridized with the REH detected in the cytoplasm without any staining of the signal with increased hybridization time, but this buffer containing the fluorescein-labelled RNA probe did not give a higher non-specific signal compared increase in non-specific signal was not a problem with 2 h hybridization and was not detected with to the standard hybridization buffer.

non-isotopic ISH. Sections hybridized with the REH buffer containing a fluorescein-labelled RNA probe at 70°C detected viral infected cells as early as 30 min DISCUSSION of hybridization whereas use of the standard hybridization buffer at 52°C only detected viral infected In this study, we have described a rapid ISH technique for the detection of nucleic acids in formalin-fixed cells after 2 h or more of hybridization. The intensity of the signal with both types of probes decreased paraffin-embedded tissues. Hybridization of either radiolabelled and fluorescein-labelled probes with with shorter hybridization time of 1 h and 30 min, but the signal was still visible. Signal intensity and the REH buffer decreased hybridization time from 20 to 2 h without compromising the intensity or the hybridization time in ISH are probably impacted by the copy number of the target nucleic acids which quality of the hybridization signal. Optimal results with strong signal and signal-to-noise ratio were ob-require at least 10 copies in a cell. 22 It may be possible to decrease hybridization time to 1 h or even 30 min, tained with both radiolabelled and fluoresceinlabelled probes by using the REH buffer at 70°C for with a higher copy number of target nucleic acid. Morphology of the cells was not impacted with either 2 h. One minor disadvantage of using the REH buffer for isotopic ISH was the slight increase in non-specific hybridization conditions is not known whether this methodology will work for the detection of DNA or with DNA probes as the incubation of samples at 90°C in a hot air oven for 15 min before hybridization with a RNA probe of 600 bond between RNA and RNA is stronger than that between DNA and DNA or DNA and RNA. Therefore, nucleotides. Musiani et al. (1994) 7 used cytospin preparations instead of formalin-fixed paraffin-em-additional studies are needed to examine potential use of the REH buffer for detecting DNA and for use bedded tissue to hybridize with a DNA probe. Both studies used higher concentrations of probes than we with DNA probes. used in this study. We used a commercially available REH and fluorescein-labelled RNA probe with an approximate size of 600 bases. The composition of

Electron microscopy of intestinal epithelial cells of chemistry 41

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of lysozyme mRNA in archival gut biopsy specimens

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Three peptidase N is a major receptor for the enteronew isolates of porcine respiratory coronavirus with pathogenic coronavirus TGEV

Sequence of the S gastroenteritis virus on the villous enterocytes of newgene from a virulent British field isolate of transmissible born pigs

Jejunal epithelium in trans-21

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In situ hybridization

veloped for membrane hybridization such as Southern

C. Beitz and B. J. Thacker for review of this article. This study was supported with a grant from the Iowa Livestock blot, northern blot, and dot blot hybridization using Health Advisory council. Senior author T. Sirinarumitr was radiolabelled DNA, RNA or oligonucleotide. The REH supported with a King's scholarship from the Anbuffer appears to be less viscous than the standard andhamahidol Foundation, Thailand. We would also like hybridization buffer. Although the mechanism of ento thank Margaret Carter at ISU Image Analysis Facility for hancement of hybridization using REH buffer is not assistance with quantitation of image signal. known, it may be due to the unfolding of viral RNA secondary structure, resulting in dissociation of protein-nucleic acid complexes so that more target REFERENCES molecules are accessible for hybridization with the probe. The lower viscosity of the REH buffer may