scholarly journals Expression of Catalase mRNA and Protein in Adult Rat Brain: Detection by Nonradioactive In Situ Hybridization with Signal Amplification by Catalyzed Reporter Deposition (ISH–CARD) and Immunohistochemistry (IHC)/Immunofluorescence (IF)

2003 ◽  
Vol 51 (6) ◽  
pp. 751-760 ◽  
Author(s):  
Arno Schad ◽  
H. Dariush Fahimi ◽  
Alfred Völkl ◽  
Eveline Baumgart
Keyword(s):  
In Situ Hybridization ◽  
Rat Brain ◽  
Mammalian Cells ◽  
Laser Scanning Microscopy ◽  
Marker Enzyme ◽  
Double Labeling ◽  
Cytoplasmic Staining ◽  
Catalyzed Reporter Deposition ◽  
Nonradioactive In Situ Hybridization

Catalase, the classical peroxisomal marker enzyme, decomposes hydrogen peroxide and is involved in the antioxidant defense mechanisms of mammalian cells. In addition, catalase can oxidize, by means of its peroxidatic activity, a variety of substrates such as methanol and ethanol, producing the corresponding aldehydes. The involvement of brain catalase in the oxidation of ethanol is well established, and severe afflictions of the CNS in hereditary peroxisomal diseases (e.g., Zellweger syndrome) are well known. Whereas the distribution of catalase in the CNS has been investigated by enzyme histochemistry and immunohistochemistry (IHC), very little is known about the exact localization of catalase mRNA in brain. Here we report the application of a tyramine/CARD (catalyzed reporter deposition)-enhanced nonradioactive in situ hybridization (ISH) protocol for detection of catalase mRNA in sections of perfusion-fixed, paraffin-embedded rat brain. Catalase mRNA could be demonstrated in a large number of neurons throughout the rat brain as a distinct cytoplasmic staining signal with excellent morphological resolution. Compared to our standard ISH protocol, the CARD-enhanced protocol for catalase mRNA detection in rat brain showed higher sensitivity and significantly better signal-to-noise ratio. In parallel IHC experiments, using an antigen retrieval method consisting of combined trypsin digestion and microwave treatment of paraffin sections, the catalase antigen was found as distinct cytoplasmic granules in most catalase mRNA-positive neurons. In addition, catalase-positive granules, presumably peroxisomes, were found by confocal laser scanning microscopy in glial cells, which were identified by double labeling immunofluorescence for GFAP and CNPase for astroglial cells and oligodentrocytes, respectively. The excellent preservation of morphology and sensitive detection of both mRNA and protein in our preparations warrant the application of the protocols described here for systematic studies of catalase and other peroxisomal proteins in diverse pathological conditions such as Alzheimer's disease and aging.

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

2000 ◽  
Vol 33 (2) ◽  
pp. 115-122 ◽  
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

scholarly journals Enzymatic Permeabilization of the Thecate Dinoflagellate Alexandrium minutum (Dinophyceae) Yields Detection of Intracellularly Associated Bacteria via Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization

2008 ◽  
Vol 74 (7) ◽  
pp. 2244-2247 ◽  
Author(s):  
Lucía Palacios ◽  
Irma Marín
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Alexandrium Minutum ◽  
Associated Bacteria ◽  
Combined Use ◽  
Catalyzed Reporter Deposition

ABSTRACT The enzymatic permeabilization procedure described here allows the detection of intracellular bacteria in the thecate dinoflagellate Alexandrium minutum by using catalyzed reporter deposition-fluorescence in situ hybridization. The combined use of propidium iodide and calcofluor for confocal laser scanning microscopy, together with general and specific fluorescent bacterial probes, demonstrated the intracellular presence of bacteria, including members of the phylum Bacteroidetes.

Catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH) detection of Dehalococcoides

2008 ◽  
Vol 73 (2) ◽  
pp. 142-147 ◽  
Author(s):  
J.A. Dijk ◽  
P. Breugelmans ◽  
J. Philips ◽  
P.J. Haest ◽  
E. Smolders ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Fish Detection ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

An in situ hybridization study on interleukin-1β mRNA induced by transient forebrain ischemia in the rat brain

1994 ◽  
Vol 26 (1-2) ◽  
pp. 135-142 ◽  
Author(s):  
Kazuki Yabuuchi ◽  
Masabumi Minami ◽  
Seishi Katsumata ◽  
Akira Yamazaki ◽  
Masamichi Satoh
Keyword(s):  
In Situ Hybridization ◽  
Rat Brain ◽  
Interleukin 1Β ◽  
Forebrain Ischemia ◽  
Hybridization Study ◽  
Transient Forebrain Ischemia

Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain

1992 ◽  
Vol 68 (3) ◽  
pp. 756-766 ◽  
Author(s):  
T. M. Perney ◽  
J. Marshall ◽  
K. A. Martin ◽  
S. Hockfield ◽  
L. K. Kaczmarek
Keyword(s):  
In Situ Hybridization ◽  
Rat Brain ◽  
K Channel ◽  
Rnase Protection ◽  
Hybridization Histochemistry ◽  
Adult Rat ◽  
In Situ Hybridization Histochemistry ◽  
Adult Rat Brain ◽  
Developing Rat

1. The gene for a mammalian Shaw K+ channel has recently been cloned and has been shown, by alternative splicing, to give rise to two different transcripts, Kv3.1 alpha and Kv3.1 beta. To determine whether these channels are associated with specific types of neurons and to determine whether or not the alternately spliced K+ channel variants are differentially expressed, we used ribonuclease (RNase) protection assays and in situ hybridization histochemistry to localize the specific subsets of neurons containing Kv3.1 alpha and Kv3.1 beta mRNAs in the adult and developing rat brain. 2. In situ hybridization histochemistry revealed a heterogeneous expression pattern of Kv3.1 alpha mRNA in the adult rat brain. Highest Kv3.1 alpha mRNA levels were expressed in the cerebellum. High levels of hybridization were also detected in the globus pallidus, subthalamus, and substantia nigra reticulata. Many thalamic nuclei, but in particular the reticular thalamic nucleus, hybridized well to Kv3.1 alpha-specific probes. A subpopulation of cells in the cortex and hippocampus, which by their distribution and number may represent interneurons, were also found to contain high levels of Kv3.1 alpha mRNA. In the brain stem, many nuclei, including the inferior colliculus and the cochlear and vestibular nuclei, also express Kv3.1 alpha mRNA. Low or undetectable levels of Kv3.1 alpha mRNA were found in the caudate-putamen, olfactory tubercle, amygdala, and hypothalamus. 3. Kv3.1 beta mRNA was also detected in the adult rat brain by both RNase protection assays and by in situ hybridization experiments. Although the beta splice variant is expressed at lower levels than the alpha species, the overall expression pattern for both mRNAs is similar, indicating that both splice variants co-expressed in the same neurons. 4. The expression of Kv3.1 alpha and Kv3.1 beta transcripts was examined throughout development. Kv3.1 alpha mRNA is detected as early as embryonic day 17 and then increases gradually until approximately postnatal day 10, when there is a large increase in the amount of Kv3.1 alpha mRNA. Interestingly, the expression of Kv3.1 beta mRNA only increases gradually during the developmental time frame examined. Densitometric measurements indicated that Kv3.1 alpha is the predominant splice variant found in neurons of the adult brain, whereas Kv3.1 beta appears to be the predominant species in embryonic and perinatal neurons. 5. Most of the neurons that express the Kv3.1 transcripts have been characterized electrophysiologically to have narrow action potentials and display high-frequency firing rates with little or no spike adaptation.(ABSTRACT TRUNCATED AT 400 WORDS)

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