scholarly journals Peptide Expression in GABAergic Neurons in Rat Suprachiasmatic Nucleus in Comparison with Other Forebrain Structures: A Double Labeling In Situ Hybridization Study

1997 ◽  
Vol 45 (9) ◽  
pp. 1231-1237 ◽  
Author(s):  
Masaki Tanaka ◽  
Tomoyuki Matsuda ◽  
Yasufumi Shigeyoshi ◽  
Yasuhiko Ibata ◽  
Hitoshi Okamura
Keyword(s):  
In Situ Hybridization ◽  
Suprachiasmatic Nucleus ◽  
Gabaergic Neurons ◽  
Hypothalamic Nucleus ◽  
Acid Decarboxylase ◽  
Expression Ratio ◽  
Double Labeling ◽  
Peptide Expression ◽  
Intermediate Part

We investigated the characteristics of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) in normal untreated rats by examination of co-expressed peptides. We adopted double labeling in situ hybridization using a digoxigenin-labeled glutamic acid decarboxylase (GAD) riboprobe and 35S-labeled peptide riboprobes. GAD mRNA-positive neurons were distributed throughout the SCN from the rostral to the caudal pole. In the dorsomedial part of the SCN, most GAD mRNA-positive neurons co-expressed arginine vasopressin mRNA. In the ventrolateral part of the SCN, about two thirds of GAD mRNA-positive neurons co-expressed vasoactive intestinal peptide (VIP) mRNA. Co-expression of GAD and somatostatin mRNA was observed in virtually all neurons of the intermediate part of the SCN. In contrast, these peptidergic traits were poorly expressed in hypothalamic GABAergic neurons outside the SCN. Vasopressin mRNA-positive cells in the supraoptic nucleus did not express GAD mRNA, and co-expression of somatostatin mRNA and GAD mRNA was rare in the periventricular hypothalamic nucleus. Similarly, the VIP mRNA co-expression ratio of GABAergic neurons in the cerebral cortex was far lower than that in the SCN.

Somatostatin neurons form a distinct peptidergic neuronal group in the rat suprachiasmatic nucleus: a double labeling in situ hybridization study

1996 ◽  
Vol 215 (2) ◽  
pp. 119-122 ◽  
Author(s):  
Masaki Tanaka ◽  
Hitoshi Okafnura ◽  
Tomoyuki Matsuda ◽  
Yasufumi Shigeyoshi ◽  
Yasuo Hisa ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Suprachiasmatic Nucleus ◽  
Hybridization Study ◽  
Double Labeling ◽  
Neuronal Group ◽  
Somatostatin Neurons

Intracellular localization of types I and II collagen mRNA and endoplasmic reticulum in embryonic corneal epithelia

1991 ◽  
Vol 100 (1) ◽  
pp. 23-33 ◽  
Author(s):  
K.K. Svoboda
Keyword(s):  
Endoplasmic Reticulum ◽  
In Situ Hybridization ◽  
Laser Scanning ◽  
Intracellular Localization ◽  
Confocal Laser ◽  
Type I ◽  
Basal Cells ◽  
Double Labeling ◽  
Collagen Mrna

The intracellular distribution of endoplasmic reticulum (ER) and types I and II collagen mRNA was analyzed in whole-mount preparations of freshly isolated corneal epithelia using in situ hybridization combined with confocal laser scanning analysis. The ER stained with DiOC6 (3) was prominent in both the periderm and basal cells. The basal cell ER distribution was perinuclear in the center of the cells, but below the nucleus the ER occupied nearly all of the cytoplasm in a reticular pattern similar to that seen with TEM cross-sections. Initial single label in situ hybridization studies showed that both the periderm and basal cells were positive for both types I and II collagen mRNA. The collagen cDNA probes appeared perinuclear in the center of the basal cells, similar to the DiOC6(3) staining pattern. In double-labeling experiments, the two mRNAs that translate chains of type I collagen, alpha 1 and alpha 2, colocalized within the same cell. However, the hybridization of probes specific for type I and II collagen mRNAs had separate, but overlapping, distributions within the same cell.

scholarly journals Expression of the c-fos protooncogene by human and murine erythroblasts

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 947-951 ◽  
Author(s):  
JF Caubet ◽  
MT Mitjavila ◽  
A Dubart ◽  
D Roten ◽  
SC Weil ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Northern Blotting ◽  
Hematopoietic Tissue ◽  
Double Labeling ◽  
Murine Bone Marrow ◽  
Marrow Cells

Abstract The expression of the c-fos protooncogene was investigated by in situ hybridization in normal murine bone marrow cells. A strong signal was found in murine marrow cells having the morphologic features of erythroblasts. This result was confirmed in human marrow cells using a double labeling technique (in situ hybridization and immunocytochemistry). A majority (70%) of the cells expressing c-fos mRNA were glycophorin A-positive. In contrast, granulocytic precursors (CD 15-positive) or monocytes and their precursors (CD 14-positive cells) did not significantly hybridize with the c-fos probe. In addition, c-fos mRNA (2.2Kb) was detected by Northern blotting in RNA extracted from homogeneous populations of erythroblasts obtained by immune panning from fetal liver and from adult blood BFU-E-derived colonies. Fos protein was also detected in erythroblasts by immunofluorescence. The high level of c-fos mRNA previously found in hematopoietic tissue should therefore be related to the transcription of the c-fos gene during terminal erythroid differentiation.

ROMK inwardly rectifying ATP-sensitive K+ channel. I. Expression in rat distal nephron segments

1995 ◽  
Vol 268 (6) ◽  
pp. F1124-F1131 ◽  
Author(s):  
W. S. Lee ◽  
S. C. Hebert
Keyword(s):  
In Situ Hybridization ◽  
Distal Tubule ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Double Labeling ◽  
Nephron Segments ◽  
Collecting Ducts ◽  
Proximal Tubular ◽  
Inwardly Rectifying

The inwardly rectifying, ATP-sensitive K+ channel (ROMK) was localized by in situ hybridization in the rat kidney. Tissue in situ hybridization revealed that transcripts encoding the ROMK channel were expressed predominantly in cortical and outer medullary nephron segments. The localization of ROMK mRNA to specific nephron segments was assessed by hybridization of isolated nephron segments with an ROMK-specific probe (single segment in situ hybridization). ROMK mRNA was present in cortical and medullary thick ascending limb, distal tubule, and cortical and outer medullary collecting ducts, but not in proximal tubule. A weak hybridization was observed with inner medullary collecting ducts. To confirm these results, serial cryosections were alternatively stained by hybridization histochemistry for ROMK mRNA or by immunocytochemistry using antibodies specific for S1, S2, or S3 proximal tubular segments. Tubular cells that displayed immunoreactivity with the proximal tubular segment-specific antibodies showed little, if any, ROMK message. In addition, using an in situ hybridization and immunocytochemistry double-labeling technique, ROMK transcripts and vitamin D-dependent calcium-binding protein were shown to colocalize to the distal tubule (distal convoluted tubule and connecting tubule). The overall nephron localization of ROMK mRNA shown in these studies is consistent with the possibility that this novel channel may represent the low-conductance ATP-sensitive K+ channel that has been identified in apical membranes of thick limb and collecting duct segments and is believed to participate in K+ secretion.

scholarly journals Autoradiographic visualization of 35S-labeled cRNA probes combined with immunoperoxidase detection of choleragenoid: a double-labeling light microscopic method for in situ hybridization and retrograde tract tracing.

1994 ◽  
Vol 42 (6) ◽  
pp. 827-831 ◽  
Author(s):  
O Hermanson ◽  
H Ericson ◽  
G Sanchez-Watts ◽  
A G Watts ◽  
A Blomqvist
Keyword(s):  
In Situ Hybridization ◽  
Cobalt Acetate ◽  
Reaction Products ◽  
Double Labeling ◽  
Cholera Toxin Subunit B ◽  
Efferent Projections ◽  
Labeled Rna ◽  
Subunit B ◽  
Silver Grains

We describe a protocol for simultaneous light microscopic visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.

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