Double immunofluorescent staining using two unconjugated primary antisera                raised in the same species.

Shindler, Kenneth S.; Roth, Kevin A.

doi:10.1177/44.11.8918908

Cited by 206 publications

(173 citation statements)

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“…Conventional immunofluorescence experiments often show good sensitivity of the method, but may give relatively high background. Immunofluorescent labelling after tyramide amplification can yield high sensitivity and low background, as reported previously by Shindler & Roth (1996).Immunofluorescence and double-labelling. Three different immunofluorescence double-labelling techniques for P2X receptor detection and cell identification were used.…”

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confidence: 75%

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Immunohistochemical identification of cells expressing ATP‐gated cation channels (P2X receptors) in the adult rat thyroid

Glaß

Burnstock

2001

Journal of Anatomy

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We carried out immunohistochemistry and western blotting of fresh frozen sections and crude extracts from adult rat thyroids. The histochemical and immunoblotting studies were performed with P2X receptor antibodies from 2 different sources. P2X-immunopositive cells were identified by fluorescence double labelling and confocal microscopy. Results of the western blotting experiments showed double bands of approximately 70 kDa and 140 kDa for all 7 P2X receptor subtypes with both sets of antibodies. Histochemical stains with antibodies from both sources also gave essentially identical results. P2X " , P2X # and P2X' receptors were detected exclusively in vascular smooth muscle ; P2X & and P2X ( receptors were also present on vascular smooth muscle. Endothelial cells stained for P2X $ , P2X % and P2X ( receptors. Thyroid follicular cells displayed immunoreactivity for P2X $ , P2X % and P2X & receptors. No immunostaining for P2X receptors was observed on C-cells. Possible roles for the broad expression of P2X receptor subtypes in the rat thyroid are discussed.Key words : Western blotting ; rat ; immunohistochemistry. Extracellular nucleotides have been shown to play an important role in cell signalling of excitable and nonexcitable cells (Abbracchio & Burnstock, 1998). Nucleotides are recognised as neurotransmitters of the central and the peripheral nervous systems (Burnstock, 1997(Burnstock, , 1999, and have been shown to be messengers in cellular growth and differentiation (Abbracchio, 1996) and to mediate apoptosis (Chow et al. 1997).Two families of receptors for extracellular nucleotides have been cloned. P2Y receptors are G proteincoupled receptors responding to purine and pyrimidine nucleotides, whereas P2X receptor are ATPgated, nonselective cation channels (Abbraccchio & Burnstock, 1994 ;Ralevic & Burnstock, 1998). The family of P2X receptors can be further subdivided into 7 subtypes, named P2X " to P2X ( (Burnstock & King, 1996 ;North & Surprenant, 2000). These P2X receptor subtypes seem to assemble to homomeric and Correspondence to Professor G. Burnstock, Autonomic Neuroscience Institute, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK. Tel. : j44 20 7830 2948 ; fax : j44 20 7830 2949 ; e-mail : g.burnstock!ucl.ac.uk heteromeric oligomers, which form the functional channel (Torres et al. 1999). The different P2X receptor homomers and heteromers were shown to have different pharmacological properties. The ability of P2X receptors to assemble to oligomers and the paucity of selective agonists and antagonists for P2X receptors make the pharmacological identification difficult. To identify the expression of P2X receptors in native tissues and cell cultures specific antibodies for all 7 P2X receptors subtypes have been generated (Oglesby et al. 1999) and recently antibodies for P2X receptors from commercial sources have also become available.In the present study we investigate the expression of P2X receptors in thyroid glands from adult rats. Th...

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confidence: 75%

“…The experiments were carried out as described by Shindler & Roth (1996). Briefly, P2X receptors were detected by tyramide amplification using P2X antibody concentrations below the detection limit of a fluorophore-coupled secondary antibody (goat antirabbit, FITC-coupled ; Nordic, NE).…”

Section: Immunohistochemistrymentioning

confidence: 99%

Immunohistochemical identification of cells expressing ATP‐gated cation channels (P2X receptors) in the adult rat thyroid

Glaß

Burnstock

2001

Journal of Anatomy

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“…Immunoreactivity was then visualized by donkey anti-rabbit conjugated to fluorescein isothiocyanate (FITC; Jackson ImmunoResearch). Because Pyk2, SAP102, and NR2A/B antibodies were raised in rabbit, tyramide signal amplification (TSA) was used for one of them, and subsequent conventional fluorescent staining was used for one of the other antibodies for double-labeling (72)(73)(74). After preincubation in 10% normal donkey serum, sections were incubated with Pyk2 antiserum at a dilution not recognized by a conventional fluorochrome-conjugated secondary antibody (1:20,000) and then reacted with biotinylated secondary antibody.…”

Section: Methodsmentioning

confidence: 99%

Interaction of the Tyrosine Kinase Pyk2 with the N-Methyl-d-aspartate Receptor Complex via the Src Homology 3 Domains of PSD-95 and SAP102

Seabold

Burette

Lim

et al. 2003

Journal of Biological Chemistry

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At low stimulus frequency, synaptic transmission depends largely upon ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) 1 -type glutamate receptors (1, 2). High frequency stimulation promotes Ca 2ϩ influx through N-methyl-D-aspartate (NMDA) receptors, thereby inducing long term potentiation (LTP) (1,3,4). LTP is a lasting increase in synaptic transmission that may underlie learning and memory (5, 6). NMDA receptors likely consist of one or two NR1 and two or three NR2 subunits; each subunit has an extracellular N terminus and an intracellular C terminus (7-10). The very C termini of NR2 subunits interact with the first two PDZ domains of PSD-95/SAP90 and the related proteins SAP102 and PSD-93/chapsyn110. PSD-95 and its homologs (SAP102, PSD-93, and SAP97) are scaffolding proteins consisting of three PDZ domains, one SH3 domain, and one GK domain. PSD-95, PSD-93, and SAP102 are thought to be involved in clustering glutamate receptors together with other proteins at postsynaptic sites (11-15). SAP97 interacts with the AMPA receptor subunit GluR1 (16 -18). SAP97 colocalizes with GluR1, but not GluR2/3, at postsynaptic sites (17). This interaction may already occur early in the secretory pathway and may be involved in trafficking or targeting of AMPA receptors (19).SH3 and GK domains of PSD-95 and its homologs form intramolecular and, to some degree, intermolecular interactions (20 -24). A point mutation in the Drosophila protein Dlg (a homolog of PSD-95) results in the substitution of a conserved leucine in the SH3 domain to a proline; this mutation leads to the loss of septate junction formation and overproliferation of the imaginal disc (25). Replacing the homologous leucine (Leu-460) with a proline in the SH3 domain of PSD-95 inhibits the interaction of the mutant SH3 domain with the GK domain (20,22). Collectively these findings indicate the physiological importance of the SH3-GK domain interaction, but a molecular function of this interaction remains to be established.Tyrosine phosphorylation of NR2B (26, 27) is augmented on LTP induction (28, 29), and several observations suggest that up-regulation of NMDA receptor activity by Src-mediated tyrosine phosphorylation is an important step in the induction of LTP. 1) Src increases NMDA receptor activity (30 -32) by elevating its peak current (33), 2) the activity of Src is up-regulated during LTP (34), 3) inhibition of Src can prevent LTP (30), and 4) an increase in synaptic transmission on Src activation occludes subsequent LTP induction (34).It is well established that Src can be activated by another tyrosine kinase, Pyk2/CAK␤/CADTK (35-38). Pyk2 is stimulated upon Ca 2ϩ influx, activation of PKC by phorbol esters, or stimulation of G q -linked receptors such as the muscarinic acetylcholine receptor M1 or the metabotropic glutamate receptor mGluR1 (35, 37, 39 -48). Neither Ca 2ϩ nor PKC appear to directly regulate Pyk2 in vitro (39); how Ca 2ϩ or PKC activates Pyk2 in vivo is unknown. When stimulated, Pyk2 autophosphorylates itself on tyrosine 402. The...

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“…For double-label immunohistochemical experiments in which both primary antibodies were raised in rabbit, the recently developed technique of "dilutional neglect" immunohistochemistry was used (Shindler and Roth, 1996). In preliminary experiments, it was determined that peripheral nerve S100␤ immunoreactivity is readily detectable with rhodamine-tyramide amplification at a primary antibody dilution of 1:10,000, whereas S100␤ immunostaining is undetectable in this same tissue with conventional immunohistochemistry when the primary antibody is diluted greater than 1:4000.…”

Section: Methodsmentioning

confidence: 99%

Expression of Neuregulins and their Putative Receptors, ErbB2 and ErbB3, Is Induced during Wallerian Degeneration

et al. 1997

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Schwann cell dedifferentiation and proliferation is a prerequisite to axonal regeneration in the injured peripheral nervous system. The neuregulin (NRG) family of growth and differentiation factors may play a particularly important role in this process, because these axon-associated molecules are potent Schwann cell mitogens and differentiation factors in vitro. We have examined Schwann cell DNA synthesis and the expression of NRGs and their receptors, the erbB membrane tyrosine kinases, in rat sciatic nerve, sensory ganglia, and spinal cord 0 -30 d postaxotomy. Analysis of NRG cDNAs from these tissues revealed several novel splice variants and showed that cells endogenous to injured nerve express NRG mRNAs. A selective induction of mRNAs encoding the glial growth factor (GGF) subfamily of NRGs occurs in nerve beginning 3 d postaxotomy and thus coincides with the onset of Schwann cell DNA synthesis. In later stages of Wallerian degeneration, however, Schwann cell mitogenesis markedly decreases, whereas elevated GGF expression persists. Of the four known erbB kinases, Schwann cells express both erbB2 and erbB3 receptors over the entire interval studied. Expression of erbB2 and erbB3 is coordinately induced in response to axotomy, indicating that Schwann cell responses to NRGs may be modulated by changes in receptor density. Neuregulin (including transmembrane precursors) and erbB protein are associated with Schwann cells postaxotomy. Thus, in contrast to the concept of NRGs as axon-associated mitogens, our findings suggest that NRGs produced by Schwann cells themselves may be partially responsible for Schwann cell proliferation during Wallerian degeneration, probably acting via autocrine or paracrine mechanisms.

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Double immunofluorescent staining using two unconjugated primary antisera raised in the same species.

Cited by 206 publications

References 21 publications

Immunohistochemical identification of cells expressing ATP‐gated cation channels (P2X receptors) in the adult rat thyroid

Immunohistochemical identification of cells expressing ATP‐gated cation channels (P2X receptors) in the adult rat thyroid

Interaction of the Tyrosine Kinase Pyk2 with the N-Methyl-d-aspartate Receptor Complex via the Src Homology 3 Domains of PSD-95 and SAP102

Expression of Neuregulins and their Putative Receptors, ErbB2 and ErbB3, Is Induced during Wallerian Degeneration

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