Zinc-Based Fixation for High-Sensitivity In Situ Hybridization: A Nonradioactive Colorimetric Method for the Detection of Rare Transcripts on Tissue Sections

2014 ◽  
pp. 125-138 ◽  
Author(s):  
Electra Stylianopoulou ◽  
George Skavdis ◽  
Maria Grigoriou
Keyword(s):  
In Situ Hybridization ◽  
Colorimetric Method ◽  
High Sensitivity ◽  
Tissue Sections

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 The random primer labeling technique applied to in situ hybridization on tissue sections.

1988 ◽  
Vol 36 (10) ◽  
pp. 1335-1340 ◽  
Author(s):  
M Thomas-Cavallin ◽  
O Aït-Ahmed
Keyword(s):  
Drosophila Melanogaster ◽  
In Situ Hybridization ◽  
Specific Activity ◽  
High Specific Activity ◽  
High Sensitivity ◽  
Random Primer ◽  
Tissue Sections ◽  
In Situ Hybridizations ◽  
Labeling Method

We report an application of the random primer labeling technique to in situ hybridizations on tissue sections. The ease of the method and the high specific activity achieved make it valuable when a large number of probes must be analyzed and high sensitivity is needed. We have applied this technique to study the spatial expression of a cluster of maternally acting genes (the yema gene region of Drosophila melanogaster which encodes eleven transcripts, some of them having a very low level of expression) (Aït-Ahmed et al., 1978: Dev Biol 122:153; Aït-Ahmed et al., unpublished results). The results reported here concern one of the transcripts of the yema region, which displays a peculiar anterior localization in the oocyte. We demonstrate that the "oligo-labeling" method allows a far better level of detection of the transcript of interest.

scholarly journals Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lenka Ulrychová ◽  
Pavel Ostašov ◽  
Marta Chanová ◽  
Michael Mareš ◽  
Martin Horn ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Schistosoma Mansoni ◽  
Rna Sequencing ◽  
Serine Proteases ◽  
Expression Patterns ◽  
High Sensitivity ◽  
Host Parasite Interactions ◽  
Highly Sensitive ◽  
Host Parasite

Abstract Background The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host–parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. Methodology Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. Results FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. Conclusions The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host–parasite interactions. Graphic abstract

High Sensitivity of PCR in Situ Hybridization for the Detection of Human Papillomavirus Infection in Uterine Cervical Neoplasias

2001 ◽  
Vol 82 (2) ◽  
pp. 350-354 ◽  
Author(s):  
Yuhong Xiao ◽  
Shigemi Sato ◽  
Takaaki Oguchi ◽  
Kaori Kudo ◽  
Yoshihito Yokoyama ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Human Papillomavirus ◽  
High Sensitivity ◽  
Human Papillomavirus Infection ◽  
Papillomavirus Infection ◽  
Pcr In Situ

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

Combined Immunohistochemical Labeling and In Situ Hybridization to Colocalize mRNA and Protein in Tissue Sections

2003 ◽  
pp. 165-176
Author(s):  
Malcolm D. Smith ◽  
Angela Parker ◽  
Riyani Wikaningrum ◽  
Mark Coleman
Keyword(s):  
In Situ Hybridization ◽  
Tissue Sections

In Situ Hybridization with Radiolabeled cRNA Probes, Using Tissue Sections and Smears

2003 ◽  
pp. 501-514
Author(s):  
Carolin Adams ◽  
Qutayla A. Hamid ◽  
Julia M. Polak
Keyword(s):  
In Situ Hybridization ◽  
Tissue Sections
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