Detection of CAIII mRNA in rat skeletal muscle and liver by in situ hybridization.

We carried out a variety of in situ methods of hybridization on rat liver and rat skeletal muscle using 35S-labeled or biotin-labeled rat carbonic anhydrase III (CAIII) cDNA clone. The methods were compared and evaluated. Use of the biotin system produced defined but nonspecific results which were shown not to be due to the biotinylated cDNA probe binding to the mRNA in the muscle sections. This artifact was shown to persist despite various attempts to eliminate it. Alternatively, using 35S-labeled cDNA gave reproducible results which were shown to be consistent with probe binding specifically to mRNA in the muscle section.     

Detection of mRNAs present at low concentrations in rat liver by in situ hybridization: application to the study of metabolic regulation and azo dye hepatocarcinogenesis

In situ hybridization on tissue sections was used to detect mRNAs present at low concentrations during metabolic adaptation and azo dye carcinogenesis in rat liver. The method consisted of hybridizing the slices at relatively high stringency with [35S]-labeled single-stranded probes derived from cDNA insert clones into the M13 phage. L-pyruvate kinase mRNA was proved to be present at very low concentrations in hepatocytes of fasted rats and to be relatively abundant in all hepatocytes after 18 hr of refeeding on a carbohydrate-rich diet. Aldolase A mRNA concentrations have been previously shown to increase markedly in liver of 3'-methyl DAB-fed rats, with a maximum at the fourth week. We demonstrate here, using our in situ hybridization technique, that this phenomenon is not due to re-expression of this "fetal marker" in hepatocytes but to its abundancy in proliferating small cells (i.e., so-called oval and transitional cells). Small amounts were also detected in sinusoidal cells. In normal liver, aldolase A mRNAs were detected only in some sinusoidal cells.     

Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization.

In this study we evaluated whether storing non-deparaffinized sections can affect the detection of specific mRNAs by radioactive in situ hybridization (ISH). Using a standard ISH protocol, we hybridized serial sections of paraffin blocks stored for different periods of time with (33)P-labeled riboprobes specific for rat Type III collagen and matrix metalloproteinase-2 (MMP-2). Signal intensities were evaluated using a phosphorimager and by blinded microscopic examination. For slides hybridized with the Type III collagen riboprobe, signal intensities measured with the phosphorimager or evaluated by microscopic examination were negatively correlated with the storage period of the sections. For slides hybridized with the MMP-2 riboprobe, differences in signal intensity could be detected, albeit inconsistently, with the phosphorimager, although microscopic examination consistently indicated stronger signals in freshly sectioned slides compared to slides stored for 2 weeks or more. We concluded that it was preferable to use recently prepared sections for trying to locate mRNAs in paraffin-embedded tissues by ISH. In addition, our results suggest that quantifying signal intensity using a phosphorimager is feasible for abundant mRNAs or when large differences in expression are anticipated.(J Histochem Cytochem 49:927-928, 2001)     

In situ hybridization of albumin mRNA in normal liver and liver tumors: Identification of hepatocellular origin

In situ hybridization was performed to detect albumin mRNA in normal liver, liver cirrhosis, primary liver tumors and secondary liver neoplasms. In areas of normal liver, and liver cirrhosis, signals for albumin mRNA were present in hepatocytes, whereas no signals were seen in other cells such as endothelial and Küpffer cells, bile duct epithelium and smooth muscle cells. In 53 of 56 hepatocellular carcinomas signals were present in tumor cells but in eight cholangiocarcinomas and 14 metastatic adenocarcinomas from large bowel or pancreas, carcinoma cells were negative for albumin mRNA. In three metastatic tumors (from two neuroendocrine carcinomas and one gastric leiomyosarcoma), tumor cells contained no signals, while the surrounding hepatocytes showed diffuse grains. In 15 of the 84 specimens examined in situ hybridization was applied to routine formalin-fixed and paraffin-embedded blocks and strong signals were obtained for albumin mRNA. We conclude that in situ hybridization of human albumin is a valid tool in the differential diagnosis of hepatocellular carcinoma from cholangiocarcinomas and tumors metastatic to the liver.     

Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate-lysine-2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the ultrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside-induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes. Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0.1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes. The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.     

Fluorescence in situ hybridization in paraffin tissue sections: pretreatment protocol

Fluorescence in situ hybridization has found wide application in the enumeration of gene and chromosome copy number both in isolated cells and in tissue sections. However, the technique has been less widely used than would be expected in formalin-fixed paraffin processed (archival) tissue. This article describes a method for assessing archival tissue sections, following pretreatment, before applying DNA probes, that gives consistent, reliable results.     

Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization.

We have adapted a non-radioactive technique to detect localized mRNAs in whole-mount Xenopus embryos. Synthetic antisense RNA transcribed in the presence of digoxygenin-UTP is used as a probe and is detected via an anti-digoxygenin antibody. We show that localized mRNAs can be detected from late gastrula to tadpole stages and that high as well as low abundance RNAs can be detected. The method was tested on muscle actin and alpha-globin RNAs, whose localization has previously been characterized. In addition, we used the method to determine the distribution of XA-1 RNA, an anterior ectoderm-specific RNA, which we show is expressed in the periphery of the cement gland as well as in the region of the hatching gland. The sequence of an XA-1 cDNA predicts a protein rich in proline and histidine.     

Detection of viral DNA and RNA by in situ hybridization

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.     

Detection and differentiation of chlamydiae by fluorescence in situ hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and PARACHLAMYDIA: The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Differential expression of type 2 and type 3 inositol 1,4,5-trisphosphate receptor mRNAs in various mouse tissues: in situ hybridization study

The inositol 1,4,5-trisphosphate receptor (IP₃R) is an intracellular Ca²⁺ release channel responsible for mobilizing stored Ca²⁺. Three different receptor types have been molecularly cloned, and their genes have been classified into a family. The gene for the type 1 receptor (IP₃R1) is predominantly expressed in cerebellar Purkinje neurons, but its gene product is localized widely in a variety of tissues; however, there is little information on what types of cells express the other two receptor types, type 2 and type 3 (IP₃R2 and IP₃R3, respectively). We studied the expression of the IP₃R gene family in various mouse tissues by in situ hybridization histochemistry. Compared with IP₃R1, the levels of expression of IP₃R2 and IP₃R3 mRNAs were low in all of the tissues tested. IP₃R2 mRNA was localized in the intralobular duct cells of the submandibular gland, the urinary tubule cells of the kidney, the epithelial cells of epididymal ducts and the follicular granulosa cells of the ovary, while the IP₃R3 mRNA was distributed in gastric cells, salivary and pancreatic acinar cells and the epithelium of the small intestine. All of these cells which express either IP₃R2 or IP₃R3 mRNA are known to have a secretory function in which IP₃/Ca²⁺ signalling has been shown to be involved, and thus either IP₃R2 or IP₃R3 may be a prerequisite to secretion in these cells.     

Localization and quantitation of opsin and transducin mRNAs in bovine retina by in situ hybridization histochemistry

Oligodeoxynucleotide probes complementary to a portion of bovine opsin mRNA and transducin mRNA were used for in situ hybridization histochemistry. Within the retina, only photoreceptors expressed mRNAs detectable with these probes, and the majority of both mRNAs were in photoreceptor inner segments. More opsin mRNA was detected than transducin mRNA. In the inner segments 0.54 ± 0.05 copies/μm³ of opsin mRNA and 0.34 ± 0.05 copies/μm³ of transducin mRNA were detected. In the outer nuclear layer, 0.39 ± 0.06 copies/ μm³ of opsin mRNA and 0.27 ± 0.04 copies/μm³ of transducin mRNA were detected.     

In situ hybridization: use of 35S-labeled probes on paraffin tissue sections

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.     

Changes in collagen and albumin mRNA in liver tissue of mice infected with Schistosoma mansoni as determined by in situ hybridization

We have employed in situ hybridization to evaluate the molecular mechanisms responsible for hypoalbuminemia and increased liver collagen content in murine schistosomiasis. Results were compared using a simplified method of hybridizing isolated hepatocytes from Schistosoma mansoni-infected and normal mouse liver with mouse albumin (pmalb-2) and chick pro-alpha 2(l) collagen (pCg45) probes. Whereas hepatocytes from infected mice showed significantly less albumin mRNA than hepatocytes from control, there were more grains of procollagen mRNA in hepatocytes from infected as compared with control liver. Hybridization of infected liver tissue sections with the collagen probe showed more grains per field in granulomas than in liver regions, whereas with the albumin probe there was more hybridization in liver tissue than in granulomas. These results suggest that in murine schistosomiasis a reduction in albumin mRNA sequence content may be associated with decreased albumin synthesis and ultimately leads to hypoalbuminemia. In addition, although the granuloma seems to be the primary source of type I collagen synthesis, hepatocytes are also capable of synthesizing collagen, especially under fibrogenic stimulation.     

Localization and regulation of mRNAs in the nervous tissue as revealed by in situ hybridization.

1. In situ hybridization histochemistry permits the study of specific mRNAs of neuropeptides, enzymes involved in the synthesis of neurotransmitters, receptors and proteins associated with glial cells in nervous tissue. 2. The central and peripheral nervous systems are composed of heterogeneous elements and specific regulatory mechanisms occur in specific cells. 3. This review will focus on the localization and regulation of different mRNAs in the nervous system from Drosophila to human, as revealed by in situ hybridization histochemistry.