The detection of cyanobacterial cells by a non-fluorescent in situ hybridization method

Seven cyanobacterial strains (Anabaena macrospora NIER10016, Oscillatoria sp. NIER10042, Microcystis aeruginosa NIER10015, M. ichtyoblabe NIER10025 and NIER10040, M. novacekii NIER10029, and M. wesenbergii NIER10068) were tested by a non-fluorescent in situ hybridization method using two specific horseradish peroxidase--labeled oligonucleotide probes and two chromogenic substrates. This approach was shown to be appropriate for analysis of natural samples.     

Imaging individual microRNAs in single mammalian cells in situ

MicroRNAs (miRNAs) are potent negative regulators of gene expression that have been implicated in most major cellular processes. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, many fundamental questions still remain regarding miRNA function and their influence on cell cycle control. Considering recent reports on the impact of cell-to-cell fluctuations in gene expression on phenotypic diversity, it is likely that looking at the average miRNA expression of cell populations could result in the loss of important information connecting miRNA expression and cell function. Currently, however, there are no efficient techniques to quantify miRNA expression at the single-cell level. Here, a method is described for the detection of individual miRNA molecules in cancer cells using fluorescence in situ hybridization. The method combines the unique recognition properties of locked nucleic acid probes with enzyme-labeled fluorescence. Using this approach, individual miRNAs are identified as bright, photostable fluorescent spots. In this study, miR-15a was quantified in MDA-MB-231 and HeLa cells, while miR-155 was quantified in MCF-7 cells. The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.     

Fluorescence in situ hybridization method for co-localization of mRNA and GEP

Co-localization studies using green fluorescent protein (GFP) and fluorescence immunohistochemistry have become commonplace. However, co-localization studies using GFP and mRNA in situ hybridization are rare, in large part because typical in situ hybridization reaction conditions often lead to the loss of GFP fluorescence. Here, we describe a new fluorescence mRNA in situ hybridization protocol using cRNA riboprobes that leaves GFP fluorescence intact. This protocol is based on a urea-based hybridization buffer and the Tyramide Signal Amplification system. This protocol should provide researchers engaged in the use of GFP with a solid starting point for adapting their own in situ hybridization protocols.     

A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections

Background

Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels.

Methodology/Principal Findings

A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50–60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method.

Conclusions/Significance

Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.     

Arginine vasotocin mRNA revealed by in situ hybridization in bovine pineal gland cells

Arginine vasopressin (AVP) is the main antidiuretic hormone in mammals and arginine vasotocin (AVT) in submammalian vertebrates. The possibility that the genetic material encoding AVT is maintained in mammals is controversial. In this study, we investigated by radioactive in situ hybridization the possible presence of the mRNA encoding AVP and AVT, and using immunocytochemistry the presence of structures immunoreactive for AVP and AVT in the bovine pineal gland. In situ hybridization was performed by use of ³⁵S-labelled oligoprobes. Immunocytochemistry was performed using specific polyclonal rabbit antibodies and the avidin-biotin-complex method. In situ hybridization revealed positive signals for both AVP mRNA and AVT mRNA in a few cells scattered throughout the pineal body. Immunocytochemistry revealed thin AVP-immunoreactive fibres in the pineal stalk and the pineal gland. It also revealed staining of several AVT-immunoreactive nerve fibres in both the pineal stalk and the gland. In addition, polyhedral, neuron-like cell bodies from which two to three processes emerged were also AVT-immunoreactive. Thus, our investigation shows the presence of AVP/AVT-immunoreactive cellular structures in the bovine pineal gland. Our data further show the presence of mRNAs encoding both AVT and AVP. We therefore suggest that AVT mRNA is translated into an AVT-like peptide in the bovine pineal.     

Improved method for detection of cellular transcripts by in situ hybridization. Detection of virus-specific RNA in rous sarcoma virus-infected cells by in situ hybridization to cDNA

Optimal conditions for the detection of complex RNA sequences in individual cells by in situ hybridization have been determined by using in vitro cultured quail embryonic cells infected with Rous sarcoma virus and a single-stranded 3H-cDNA probe of high specific activity complementary to the RSV genome. It is shown that fixation of target tissue can be suitably achieved by using glutaraldehyde at low concentration, and subjecting cytological preparations to heat post-fixation treatment. Conditions for removing unhybridized radioactive probe molecules by means of S1 nuclease are reported.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Summary In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using³⁵S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using³⁵S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

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

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.