Topography of forming and resorbing cells on endosteal surfaces of the rabbit humerus by double-staining with in situ hybridization and tartrate-resistant acid phosphatase reaction: a new model to study the bone reaction to loading

1995 ◽  
Vol 6 (5) ◽  
pp. 279-283 ◽  
Author(s):  
C. F. Voigt ◽  
P. Peljak ◽  
C. M�ller-Mai ◽  
H. Herbst ◽  
U. M. Gross ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Acid Phosphatase ◽  
Tartrate Resistant Acid Phosphatase ◽  
Double Staining ◽  
New Model ◽  
Bone Reaction ◽  
Acid Phosphatase Reaction

Localization of the human type 5, tartrate-resistant acid phosphatase gene by in situ hybridization

Genomics ◽  
1989 ◽  
Vol 4 (4) ◽  
pp. 597-600 ◽  
Author(s):  
Beverly S. Allen ◽  
Catherine M. Ketcham ◽  
R. Michael Roberts ◽  
Harry S. Nick ◽  
Harry Ostrer
Keyword(s):  
In Situ Hybridization ◽  
Acid Phosphatase ◽  
Tartrate Resistant Acid Phosphatase ◽  
Human Type ◽  
Acid Phosphatase Gene

scholarly journals Detection of estrogen receptor alpha, carbonic anhydrase II and tartrate-resistant acid phosphatase mRNAs in putative mononuclear osteoclast precursor cells of neonatal rats by fluorescence in situ hybridization

1998 ◽  
Vol 20 (2) ◽  
pp. 211-219 ◽  
Author(s):  
WH Huang ◽  
AT Lau ◽  
LL Daniels ◽  
H Fujii ◽  
U Seydel ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
In Situ Hybridization ◽  
Acid Phosphatase ◽  
Carbonic Anhydrase ◽  
Precursor Cells ◽  
Osteoclast Precursor ◽  
Carbonic Anhydrase Ii ◽  
Tartrate Resistant Acid Phosphatase ◽  
Gene Transcripts

Increasing evidence suggests that estrogen deficiency in women promotes the expansion of populations of bone marrow cells that differentiate into osteoclasts under the influence of osteotropic hormones and local factors. A progressive cytoplasmic accumulation of osteoclastic bone resorbing enzymes, such as tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II), characterizes osteoclast differentiation. To evaluate the possibility that estrogen may have a direct effect on osteoclast precursor cells, we investigated the mRNA levels of estrogen receptor a (ERa), TRACP and CA II genes in neonatal rat bone imprints by fluorescence in situ hybridization and confocal microscopy. Morphological assessment of bone imprints has shown that the putative mononuclear osteoclast precursor cells (MOPC) display strongly basophilic cytoplasm and a low nuclear/cytoplasmic ratio, while some of these cells possess pale-staining ruffled border regions similar to those observed in osteoclasts. Both CA II and TRACP mRNAs were detected in putative MOPC as well as multinuclear osteoclasts. The gene transcripts were mainly located in the cytoplasm of these cells. To determine whether these putative MOPC possess ER mRNA, a 637 base pair antisense ER riboprobe was used. The results indicated that MOPC which show TRACP reactivity express high levels of ER gene transcripts in their cytoplasm. In contrast, only a few multinuclear osteoclasts in the bone imprints possessed ER gene transcripts. Interestingly, the levels of ER mRNA in these multinuclear osteoclasts were very low compared with those in the putative MOPC. Treatment with RNase prior to hybridization resulted in a significant loss of signal in these cells. The results of these studies suggest that estrogen may have a direct role in modulating the recruitment of osteoclast precursor cells during osteoclastogenesis.

scholarly journals Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

1990 ◽  
Vol 38 (3) ◽  
pp. 325-329 ◽  
Author(s):  
W van den Brink ◽  
C van der Loos ◽  
H Volkers ◽  
R Lauwen ◽  
F van den Berg ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Staining Method ◽  
Double Staining ◽  
Reaction Products ◽  
Silver Enhancement ◽  
Silver Staining Method ◽  
Double Staining Method ◽  
Beta Galactosidase

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

scholarly journals Human osteoclast and giant cell differentiation: the apparent switch from nonspecific esterase to tartrate resistant acid phosphatase activity coincides with the in situ expression of osteopontin mRNA.

1995 ◽  
Vol 43 (12) ◽  
pp. 1193-1201 ◽  
Author(s):  
J R Connor ◽  
R A Dodds ◽  
I E James ◽  
M Gowen
Keyword(s):  
Acid Phosphatase ◽  
Mononuclear Cells ◽  
Giant Cells ◽  
Mononuclear Phagocyte ◽  
Osteoclast Precursor ◽  
Tartrate Resistant Acid Phosphatase ◽  
Nonspecific Esterase ◽  
Connective Tissues ◽  
Human Osteoclast

Animal model and in vitro cultures suggest that osteoclasts and cells of the mononuclear phagocyte system share a common precursor. However, the human osteoclast precursor has not been positively identified. We attempted to identify the precursor in situ by using a number of osteoclast- and macrophage-selective markers, together with the expression of osteopontin mRNA, previously shown to be abundant in human osteoclasts. Sections of osteophytic bone and a panel of inflammatory connective tissues were processed for in situ hybridization; serial sections were analyzed for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE) activity, selective cytochemical markers for the osteoclast and cells of the macrophage/monocyte lineage, respectively. The murine anti-human osteoclast monoclonal antibodies 23C6 (vitronectin receptor) and C35 (osteoclast-selective) were used to further identify the osteoclast phenotype. We compared osteoclasts, giant cells, and their respective putative mononuclear precursors. At resorption sites within osteophytic bone, osteopontin mRNA was expressed in osteoclasts and a distinct population of TRAP+, NSE- mononuclear cells. Adjacent clusters of mononuclear cells were TRAP- and NSE+ or were active for both enzymes; these cells demonstrated variable expression of osteopontin mRNA. In the inflammatory connective tissues, abundant macrophage-like cells (NSE+/TRAP-) did not express osteopontin mRNA. However, TRAP+ mononuclear cells observed among clusters of NSE+ cells did express osteopontin mRNA. At these sites, clusters of putative macrophage polykaryons removing fragments of bone debris were observed. These giant cells and associated mononuclear cells were NSE- and distinctly TRAP+, and expressed osteopontin mRNA, C35, and 23C6 (human osteoclast) reactivity. Therefore, cells involved in the remodeling (resorption) of bone or the removal of bone debris, together with their immediate precursors, switch from being NSE+/TRAP- to NSE-/TRAP+ cells that express osteopontin mRNA. We propose that the clusters of NSE+/TRAP- mononuclear cells represent the immature osteoclast precursor. In support of this, TRAP+/NSE+ cells were occasionally observed in both tissues, representing an intermediate stage in differentiation. These results further suggest that cells of the mononuclear phagocyte lineage within bone and inflammatory connective tissue have the potential to differentiate into osteoclasts.

A Double Staining Method for Histone H3 mRNA and p53 Protein in Oral Tumors Using In Situ Hybridization and Immunohistochemistry.

1999 ◽  
Vol 32 (4) ◽  
pp. 327-332 ◽  
Author(s):  
Tetsunari Nishikawa ◽  
Shoichi Arai ◽  
Kenichi Uobe ◽  
Masahiro Wato ◽  
Kazuya Tominaga ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Staining Method ◽  
P53 Protein ◽  
Histone H3 ◽  
Double Staining ◽  
Double Staining Method ◽  
Oral Tumors

Four-Hour Double Staining for In Situ Hybridization and Immunohistochemistry

2000 ◽  
Vol 23 (4) ◽  
pp. 321-325 ◽  
Author(s):  
Benio Tsuchiya ◽  
Yuichi Sato ◽  
Kathleen T. Montone ◽  
Tatsuo Nagai ◽  
Toru Kameya
Keyword(s):  
In Situ Hybridization ◽  
Double Staining

scholarly journals Osteoclastic Tartrate-resistant Acid Phosphatase (Acp 5): Its Localization to Dendritic Cells and Diverse Murine Tissues

2000 ◽  
Vol 48 (2) ◽  
pp. 219-227 ◽  
Author(s):  
Alison R. Hayman ◽  
Alison J. Bune ◽  
John R. Bradley ◽  
Jeremy Rashbass ◽  
Timothy M. Cox
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Acid Phosphatase ◽  
Histochemical Staining ◽  
Tartrate Resistant Acid Phosphatase ◽  
Fluorescence Studies ◽  
Tissue Sections ◽  
Adult Mice ◽  
Mouse Tissues

Tartrate-resistant acid phosphatase (TRAP) is a histochemical marker of the osteoclast. It is also characteristic of monohistiocytes, particularly alveolar macrophages, and is associated with diverse pathological conditions, including hairy cell leukemia and AIDS encephalopathy. To study the biology of this enzyme, we investigated its expression and activity in mouse tissues. Confocal fluorescence studies showed that TRAP is localized to the lysosomal compartment of macrophages. In adult mice, high activities of the enzyme were demonstrated in bone, spleen, liver, thymus, and colon, with lower amounts in lung, stomach, skin, brain, and kidney. Trace amounts were detected in testis, muscle, and heart. Expression of TRAP mRNA was investigated in tissue sections by in situ hybridization and protein expression was monitored by histochemical staining or immunohistochemically. TRAP is widely expressed in many tissues, where it is associated with cells principally originating from the bone marrow, including those of osteoclast/macrophage lineage. The cellular distribution of TRAP mRNA and enzyme antigen in the tissues corresponds closely to that of cells staining with an antibody directed to the CD80 (B7) antigen. Therefore, to confirm its putative localization in dendritic cells, isolated bone marrow dendritic cells were matured in culture. These co-stained strongly for TRAP protein and the CD80 antigen. These studies demonstrate that TRAP is a lysosomal enzyme that is found in diverse murine tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages, as previously shown.

scholarly journals Sensitive detection of rat gastrin mRNA by in situ hybridization with chemically biotinylated oligodeoxynucleotides: validation, quantitation, and double-staining studies.

1993 ◽  
Vol 41 (2) ◽  
pp. 157-163 ◽  
Author(s):  
L I Larsson ◽  
D M Hougaard
Keyword(s):  
In Situ Hybridization ◽  
Detection System ◽  
Northern Blotting ◽  
Model Systems ◽  
Double Staining ◽  
Adult Rats ◽  
Gastrin Cells ◽  
Human Gastrin ◽  
Almost All

Chemically biotin-labeled oligonucleotides form attractive reagents, as large quantities of stable and well-defined probes can easily be produced. Their usefulness for in situ hybridization was tested using rat gastrin cells as a model. Two probes recognizing two different regions of rat gastrin mRNA were synthesized and produced specific and equally strong hybridization signals. A probe complementary to human gastrin mRNA, but with mismatches to the rat gastrin mRNA sequence, failed to reveal rat gastrin cells under the stringency conditions used. Northern blotting revealed that the rat gastrin mRNA probes reacted exclusively with the appropriately sized (approximately 650 bases) mRNA. Model systems demonstrated that the hybridization signal, as revealed by alkaline phosphatase-based detection, varied linearly with the 10logarithm of target concentration and also showed that a new detection system was much more sensitive than previously used systems. In agreement with previous biochemical data, image analysis showed that starvation of rats led to a progressive decrease in cell staining intensities and cell numbers. Double staining for rat gastrin mRNA and gastrin immunoreactivity showed that in adult rats almost all gastrin cells expressed both mRNA and protein. Similar studies on developing rat gastrin cells revealed discrepancies between gastrin mRNA and gastrin-immunoreactive cells during the first week of newborn life. Subsequently, expression of mRNA and protein in the cells became gradually more concordant.

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