Immunofluorescence Labeling Method Research Articles

Organization of enkephalinergic neuronal system in the central nervous system of the gecko Hemidactylus frenatus.

Enkephalins are endogenous opioid pentapeptides that play a role in neurotransmission and pain modulation in vertebrates. However, the distribution pattern of enkephalinergic neurons in the brains of reptiles has been understudied. This study reports the organization of the methionine-enkephalin (M-ENK) and leucine-enkephalin (L-ENK) neuronal systems in the central nervous system of the gecko Hemidactylus frenatus using an immunofluorescence labeling method. Although M-ENK and L-ENK-immunoreactive (ir) fibers extended throughout the pallial and subpallial subdivisions, including the olfactory bulbs, M-ENK and L-ENK-ir cells were found only in the dorsal septal nucleus. Enkephalinergic perikarya and fibers were highly concentrated in the periventricular and lateral preoptic areas, as well as in the anterior and lateral subdivisions of the hypothalamus, while enkephalinergic innervation was observed in the hypothalamic periventricular nucleus, infundibular recess nucleus and median eminence. The dense accumulation of enkephalinergic content was noticed in the pars distalis of the hypophysis. In the thalamus, the nucleus rotundus and the dorsolateral, medial, and medial posterior thalamic nuclei contained M-ENK and L-ENK-ir fibers, whereas clusters of M-ENK and L-ENK-ir neurons were observed in the pretectum, mesencephalon, and rhombencephalon. The enkephalinergic fibers were also seen in the area X around the central canal, as well as the dorsal and ventral horns. The widespread distribution of enkephalin-containing neurons within the central nervous system implies that enkephalins regulate a variety of functions in the gecko, including sensory, behavioral, hypophysiotropic, and neuroendocrine functions.

In Vivo Study on the Effect of Tetrahydropalmatine on the Activation of NLRP3 Inflammasome in BV-2 Cells

Background: The paper aimed to explore the effect of Tetrahydropalmatine on the activation of NLRP3 inflammasomes in BV-2 cells and its mechanism in vitro. Material and methods: ELISA determine the IL-1β and IL-18 in the BV-2 cell culture medium of each group. WB was employed to detect NLRP3 inflammasome-related components including NLRP3, ASC, NEK7 and Caspase-1, as well as p-NF-κB, NF-κB, p-Iκ-Bα and Iκ-Bα in cell lysates of each group. The effects of Tetrahydropalmatine on NLRP3 inflammasome activation and NF-κB signaling pathway were analyzed. Immunofluorescence labeling method was used to detect the expressions of NLRP3 in BV-2 cells in each group. Mito-tracker Red labeled mitochondrial confocal microscope was employed to observe and evaluate mitochondrial damage. Fluorescent probe DCFH-DA was used to label cells. Flow cytometry technology was applied to detect ROS production and evaluate the effect of Tetrahydropalmatine on ROS production. Results: After Tetrahydropalmatine treatment, p-NF-κB/NF-κB and p-Iκ-Bα/Iκ-Bα were significantly lower than that in the model group. Tetrahydropalmatine can inhibit the production of ROS and improve the mitochondrial membrane potential. Conclusion: Tetrahydropalmatine can mitigate mitochondrial damage, reduce ROS production, and inhibit the NF-κB signaling pathway, thereby inhibiting the activation of NLRP3 inflammasomes in BV-2 cells.

Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord

BackgroundThe use of morphine in clinical medicine is severely constrained by tolerance. Therefore, it is essential to examine pharmacological therapies that suppress the development of morphine tolerance. Amiloride suppressed the expression of inflammatory cytokines by inhibiting microglial activation. Microglia play a crucial role in the establishment of morphine tolerance. Thus, we anticipated that amiloride might suppress the development of morphine tolerance. During this investigation, we assessed the impact of amiloride on mouse morphine tolerance. MethodsMice received morphine (10 mg/kg, s.c.) twice daily with intrathecally injected amiloride (0.3 μg/5 μl, 1 μg/5 μl, and 3 μg/5 μl) for nine continuous days. To assess morphine tolerance, mice underwent the tail-flick and hot plate tests. BV-2 cells were used to investigate the mechanism of amiloride. By using Western blotting, real-time PCR, and immunofluorescence labeling methods, the levels of acid-sensing ion channels (ASICs), nuclear factor kappa B (NF-kB) p65, p38 mitogen-activated protein kinase (MAPK) proteins, and neuroinflammation-related cytokines were determined. ResultsThe levels of ASIC3 in the spinal cord were considerably increased after long-term morphine administration. Amiloride was found to delay the development of tolerance to chronic morphine assessed via tail-flick and hot plate tests. Amiloride reduced microglial activation and downregulated the cytokines IL-1β and TNF-a by inhibiting ASIC3 in response to morphine. Furthermore, amiloride reduced p38 MAPK phosphorylation and inhibited NF-κB expression. ConclusionsAmiloride effectively reduces chronic morphine tolerance by suppressing microglial activation caused by morphine by inhibiting ASIC3.

Changes in Homogalacturonan Metabolism in Banana Peel during Fruit Development and Ripening.

Though numerous studies have focused on the cell wall disassembly of bananas during the ripening process, the modification of homogalacturonan (HG) during fruit development remains exclusive. To better understand the role of HGs in controlling banana fruit growth and ripening, RNA-Seq, qPCR, immunofluorescence labeling, and biochemical methods were employed to reveal their dynamic changes in banana peels during these processes. Most HG-modifying genes in banana peels showed a decline in expression during fruit development. Four polygalacturonase and three pectin acetylesterases showing higher expression levels at later developmental stages than earlier ones might be related to fruit expansion. Six out of the 10 top genes in the Core Enrichment Gene Set were HG degradation genes, and all were upregulated after softening, paralleled to the significant increase in HG degradation enzyme activities, decline in peel firmness, and the epitope levels of 2F4, CCRC-M38, JIM7, and LM18 antibodies. Most differentially expressed alpha-1,4-galacturonosyltransferases were upregulated by ethylene treatment, suggesting active HG biosynthesis during the fruit softening process. The epitope level of the CCRC-M38 antibody was positively correlated to the firmness of banana peel during fruit development and ripening. These results have provided new insights into the role of cell wall HGs in fruit development and ripening.

Effect of pretreatment of acupuncture on learning-memory ability and related protein of NLRP3 inflammasome in hippocampus in Alzheimer's disease like rats

To observe the effect of pretreatment of acupuncture on the expression of nucleotide-binding oligomerization domain-like receptor 3(NLRP3), Caspase-1, interleukin1β(IL-1β) and the number of activated microglia (MG) in the hippocampus in Alzheimer's disease (AD) like rats, so as to explore the mechanism of pretreatment of acupuncture in preventing and treating AD. A total of 36 SD rats were randomly divided into a blank group, a model group and an electroacupuncture (EA) group, 12 rats in each group. The AD like rat model was established by 8-week continuous intraperitoneal injection of D-galactose (120 mg·kg-1·d-1) in the model group and the EA group. The rats in the EA group were intervened with EA at "Baihui" (GV 20) and "Zusanli" (ST 36), continuous wave, 5 Hz in frequency, 1 mA in intensity, 20 min each intervention, once a day, for 8 weeks. After the intervention, the spatial learning-memory ability was evaluated by the Morris water maze test. The expression levels of NLRP3, Caspase-1 and IL-1β protein in hippocampus were detected by Western blot method. The number of activated MG in hippocampus was detected by immunofluorescence labeling method. Compared with the blank group, the average escape latency was prolonged (P<0.01), the number of platform crossing was reduced (P<0.01), and the exploration time of target quadrant was shortened (P<0.01) in the model group. Compared with the model group, the average escape latency was shortened (P<0.01), the number of platform crossing was increased (P<0.01), and the exploration time of target quadrant was prolonged (P<0.01) in the EA group. Compared with the blank group, the expression levels of NLRP3, Caspase-1, IL-1β protein in hippocampus were increased (P<0.01), and the number of activated MG was increased (P<0.01) in the model group. Compared with the model group, the expression levels of NLRP3, Caspase-1, IL-1β protein in hippocampus were decreased (P<0.01), and the number of activated MG was decreased (P<0.05) in the EA group. Pretreatment of acupuncture could prevent and treat the learning-memory dysfunction in AD like rats, and its mechanism may be related to the inhibition of NLRP3 inflammatsome related protein and MG activation.

Acceleration of Carbon Fixation in Chilling-Sensitive Banana under Mild and Moderate Chilling Stresses.

Banana is one of the most important food and fruit crops in the world and its growth is ceasing at 10–17 °C. However, the mechanisms determining the tolerance of banana to mild (>15 °C) and moderate chilling (10–15 °C) are elusive. Furthermore, the biochemical controls over the photosynthesis in tropical plant species at low temperatures above 10 °C is not well understood. The purpose of this research was to reveal the response of chilling-sensitive banana to mild (16 °C) and moderate chilling stress (10 °C) at the molecular (transcripts, proteins) and physiological levels. The results showed different transcriptome responses between mild and moderate chilling stresses, especially in pathways of plant hormone signal transduction, ABC transporters, ubiquinone, and other terpenoid-quinone biosynthesis. Interestingly, functions related to carbon fixation were assigned preferentially to upregulated genes/proteins, while photosynthesis and photosynthesis-antenna proteins were downregulated at 10 °C, as revealed by both digital gene expression and proteomic analysis. These results were confirmed by qPCR and immunofluorescence labeling methods. Conclusion: Banana responded to the mild chilling stress dramatically at the molecular level. To compensate for the decreased photosynthesis efficiency caused by mild and moderate chilling stresses, banana accelerated its carbon fixation, mainly through upregulation of phosphoenolpyruvate carboxylases.

Cruciform DNA in mouse growing oocytes: Its dynamics and its relationship with DNA transcription

Cruciform DNA is a causing factor of genome instability and chromosomal translocation, however, most studies about cruciform DNA in mammalian cells were based on palindromic sequences containing plasmids and reports about endogenous cruciform DNA are rare. In this study we observed the dynamics of endogenous cruciform DNA in mouse growing oocytes using immunofluorescence labeling method. We found cruciform DNA foci exist in transcription active growing oocytes but not in transcription inactive fully grown oocytes and colocalized with Parp1 but not with DNA damage marker γH2A.X. By analyzing the Genotype-Tissue Expression data, we found cruciform DNA-mediated chromosomal translocation in human spermatocytes is associated with the specific DNA transcription in testis. When inhibiting the transcription with α-amanitin in mouse oocytes, we found oocyte cruciform DNA foci decreased significantly. In summary, we observed the endogenous cruciform DNA in growing oocytes and our results showed that the cruciform DNA formation is transcription-dependent.

Antiepileptic effects of exogenous β-hydroxybutyrate on kainic acid-induced epilepsy.

The aim of the present study was to explore the potential anticonvulsant effects of β-hydroxybutyrate (BHB) in a kainic acid (KA)-induced rat epilepsy model. The KA-induced rat seizure model was established and BHB was administrated intraperitoneally at a dose of 4 mmol/kg 30 min prior to KA injection. Hippocampal tissues were then obtained 1, 3 and 7 days following KA administration, following which the expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were measured using a double immunofluorescence labeling method. In addition, the contents of glutathione (GSH), γ-aminobutyric acid (GABA) and ATP were measured using ELISA. Pretreatment with BHB markedly increased the expression of NSE after KA injection compared with that in the normal saline (NS) + KA group, suggesting that the application of BHB could alleviate neuronal damage in rats. The protective effect of BHB may be associated with suppressed inflammatory responses, which was indicated by the observed inhibition of GFAP expression in rats in the BHB + KA group compared with that in the NS + KA group. It was also found that GSH and GABA contents were notably increased after the rats were pretreated with BHB compared with those in the NS + KA group. To conclude, the application of exogenous BHB can serve as a novel therapeutic agent for epilepsy.

Cranberry Juice Phytochemicals Attenuated Ethanol-Induce Epithelial Barrier Dysfunction on Caco-2 Monolayers (P06-094-19)

ObjectivesThe objective of this study was to determine whether cranberry juice phytochemicals protect intestinal barrier using Caco-2 monolayer models, and to elucidate the underlying mechanisms of cranberry juice phytochemicals on ethanol-induced barrier dysfunction. MethodsCranberry juice phytochemicals were extracted using Amberlite FPX66 resin and phytochemicals composition were analyzed on HPLC. Caco-2 monolayers were incubated with cranberry juice phytochemicals at different concentrations for 2 hours before treatment with 4.5% ethanol. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-labeled dextran (FITC) was used to evaluate paracellular permeability. Epithelial integrity and distribution of tight junction proteins (Occludin and ZO-1) in Caco-2 monolayers were determined by immunofluorescence labeling method. Protein levels of ZO-1, Occludin and Claudin-1 were determined by immunoblotting. ResultsCranberry juice phytochemicals consisted of malvidin 3,5-diglucoside, petunidin 3-glucoside, chlorogenic acid and caffeic acid. Ethanol induced decrease of TEER while pretreatment of cranberry juice phytochemicals protected Caco-2 monolayer integrity in a dose-dependent manner. Monolayers treated with 7.5, 15, and 30 μg/mL of cranberry juice phytochemicals had TEER value 11.6%, 22.7% and 30.5% higher than the ethanol-treatment monolayers, respectively. This was accompanied by 40%, 55%, and 50% decreases of the paracellular permeability compared to ethanol-treated monolayers. Immunofluorescent analysis showed that occludin was localized tightly around cell membrane and colocalize with ZO-1 in the normal Caco-2 monolayers. Exposure to 4.5% ethanol disrupted both occludin and ZO-1 networks and resulted in gap formation among the cells. Cranberry juice phytochemicals attenuated these changes induced by ethanol exposure. Ethanol exposure significantly reduced the protein levels of occludin and ZO-1. Pretreatment with cranberry juice phytochemicals increased the protein levels of occludin dose dependently but did not affect ZO-1 level. ConclusionsCurrent study suggested that cranberry juices phytochemicals attenuate ethanol-induced intestinal barrier dysfunction in part by enhancing the tight junction assemblies. Funding SourcesNA.

Elevated galanin receptor type 2 primarily contributes to mechanical hypersensitivity after median nerve injury.

In this study, we investigated temporal changes in galanin receptor type 2 (GalR2) expression in NF200-, galanin-, neuropeptide Y (NPY)-, and neuronal nitric oxide synthase (nNOS)-like immunoreactive (LI) dorsal root ganglion (DRG) neurons after median nerve chronic constriction injury (CCI), and the effects of GalR2 on c-Fos expression in the cuneate nucleus (CN). Double immunofluorescence labeling methods were used to appraise changes in GalR2 expression in NF200-LI, galanin-LI, NPY-LI, and nNOS-LI DRG neurons after CCI. The von Frey assay was used to assess the efficiency of intraplantar administration of saline, M871 (a GalR2 antagonist), or AR-M1896 (a GalR2 agonist) on neuropathic signs of rats with CCI. The effects of alterations in c-Fos expression were assessed in all treatments. The percentage of GalR2-LI neurons in lesioned DRGs increased and peaked at 1 week after CCI. We further detected that percentages of GalR2-LI neurons labeled for NF200, galanin, NPY, and nNOS significantly increased following CCI. Furthermore, M871 remarkably attenuated tactile allodynia, but the sensation was slightly aggravated by AR-M1896 after CCI. Consequentially, after electrical stimulation of the CCI-treated median nerve, the number of c-Fos-LI neurons in the cuneate nucleus (CN) was significantly reduced in the M871 group, whereas it increased in the AR-M1896 group. These results suggest that activation of GalR2, probably through NPY or nitric oxide, induces c-Fos expression in the CN and transmits mechanical allodynia sensations to the thalamus.

Porcine IGF1 synonymous mutation alter gene expression and protein binding affinity with IGF1R

ContextInsulin like growth factor 1 (IGF1) is privotal in the regulation of animal growth and is a single-chain globular protein composed of B, C, A, and D regions, of which the C region is involved in maintaining high affinity binding to the IGF1 receptor (IGF1R). PurposeIn this study, significant expression differences between large pigs and miniature pigs were detected and only one synonymous SNP (c.258G>A) in the C region of the coding sequence of IGF1 gene was screened. The aim of this manuscript was to clear the function of the SNP and clarify the mechanism of its influnce. MethodsThe expression vectors contained A allele and G allele were constructed, and the expression assays of the two groups were determined by qRT-PCR and western blotting, then the stability assays of the mRNA and protein were carried out under the inhibitation of actinomycin D and cycloheximide, respectively. At last, the binding affinity of IGF1-G and IGF1-A with IGF1R were indicated by co-immunoprecipitation and double immunofluorescence labeling methods, the conformation difference was detected by differential immunodetection. ResultsThe IGF1-G expressed higher than IGF1-A in both transcription and translation levels, and the mRNA and protein stabilities of IGF1-G were lower than IGF1-A (P < 0.05). Furthermore, the relative binding affinity of GG-genotype IGF1 with IGF1R was significantly higher than that of the AA-genotype IGF1 (P < 0.05), and there was a difference in the conformation of the IGF1 with two genotypes. ConclusionOur findings indicated the synonymous mutation altered the IGF1 gene expression and confirmed the synonymous mutation affected the IGF1 folding and the interactions with the IGF1R preliminarily.

Mechanisms of autophagy and apoptosis mediated by JAK2 signaling pathway after spinal cord injury of rats.

The aim of this study was to investigate the pathogenesis of autophagy and apoptosis mediated by Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal pathway after the onset of acute spinal cord injury (ASCI). A total of 45 Sprague-Dawley adult rats of either sex were selected for this study. The age of rats ranged from 8 to 10 weeks, and the average weight was 245 g. These rats were randomly divided into three groups, i.e. sham-operated group, model group, and the AG-490 intervention group (AG-490 is an inhibitor of JAK2). Each group contained 15 rats. Models were prepared using the modified Allen method. Five rats in each group were sacrificed at 6, 12 and 24 h, respectively, and the expression levels of p-JAK2 and p-STAT3 were detected in spinal cord tissue via western blot analysis. The levels of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected via ELISA, positive expression of light chain 3 (LC3)-II of microtubule-associated protein 1 via immunofluorescence labeling method, and mRNA expression levels of caspase-3 and Bax/Bcl-2 via RT-PCR. In the model group, the expression levels of p-JAK2, p-STAT3, IL-6, TNF-α and LC3-II, and the mRNA expression levels of caspase-3 and Bax/Bcl-2 at all time-points were significantly higher than those in the AG-490 intervention group, and the levels in the sham-operated group were the lowest (p<0.05). In the model group, peak levels of p-JAK2 and p-STAT3 were attained at 12 h, but a decline was seen at 24 h; while increasing trend was seen in other indicators. In conclusion, JAK2/STAT3 signal pathway can mediate the activity of autophagy and apoptosis in an early stage after the onset of ASCI of rat.

Immunohistochemical characterization of the jugular (superior vagal) ganglion in the pig.

The study was carried out on three 4-month old female pigs. All the animals were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde (pH 7.4). Left and right superior vagal ganglia (SVG) were collected and processed for immunofluorescence labeling method. The preparations were examined under a Zeiss LSM 710 confocal microscope equipped with adequate filter block. Neurons forming SVG were round or oval in shape with a round nucleus in the center. The majority of them (52%) were medium (M) (31-50 μm in diameter) while 7% and 41% were small (S) (up to 30μm in diameter) or large (L) (above 50 μm in diameter) in size, respectively. Double-labeling immunofluorescence revealed that SVG neurons stained for CGRP (approx. 57%; among them 37%, 9% and 54% were M, S and L in size, respectively), SP (14.5%; 72.4% M, 3.4% S, 24.2% L), VACHT (26%; 63% M, 24% S and 13% L), GAL (14%; 57% M, 29% S, 14% L), NPY (12%; 53% M, 12% S, 35% L), Met-Enk (5%; 40% M, 6% S and 54% L), PACAP (15%; 52% M, 24% S and 24% L), VIP (6.3%; 67% M, 8% S and 25% L), and NOS-positive (6%; 31% M and 69% L). The most abundant populations of intraganglionic nerve fibers were those which stained for CGRP or GAL, whereas only single SP-, PACAP- or Met-ENK-positive nerve terminals were observed.

Molecular mechanism of P75NTR gene-induced apoptosis in tongue squamous cell carcinoma cell

Objective To investigate the molecular mechanism of P75NTR gene-induced apoptosis in tongue squamous cell carcinoma Tca8113 cell lineage. Methods P75NTR specific siRNA was transferred into P75NTR positive tongue squamous cell carcinoma Tca8113 cells. P75NTR positive Tca8113 cells were divided into 4 groups: blank group (without transfection), negative control group (transfected with negative control siRNA), experiment group-776 (transfected with siRNA-P75NTR-776) and experiment group-1234 (transfected with siRNA-P75NTR-1234). Transfection efficiency and cell apoptosis were detected by flow cytometry. The interference effect of P75NTR mRNA expression was detected by fluorescence quantitative PCR. 3-(4, 5-dimethyl-2-thiazoly)-2, 5-diphenyl-2H-tetrazolium bromide assay was applied in measuring cell prolife-ration. The protein changes of P75NTR were detected by Western blotting. The distributions of nuclear factor-κB(NF-κB) of cells were observed by cell immunofluorescence labeling method. Results The transfection efficiency was 30%. The apoptosis rate of experiment group-776, experiment group-1234 and negative control group was (20.35±0.18) %, (12.32±1.51)% and (2.63±0.10)% respectively. Compared with the negative control group, the differences of the former two group had statistical significance (t=177.20, P<0.005; t=37.12, P<0.005). The P75NTR gene interference was successful. The inhibition rate of P75NTR protein reached 31% in experiment group-776. The cell viability of Tca8113 cells after P75NTR-siRNA interference was 70.02%, 78.01% and 95.81% in experiment group-776, experiment group-1234 and negative control group. And there were significant differences between experiment group-776 and negative control group(χ2= 235.3, P<0.010), and between experiment group-1234 and negative control group (χ2= 117.5, P<0.005). NF-κB distribution was increased in cell cytoplasm in the interference group than that in control group. Conclusion P75NTR may promote the proliferation or inhibit the apoptosis of tongue squamous cell carcinoma, and the molecular mechanism may be correlated with hindering the transportion of NF-κB into cell nuclear. Key words: Apoptosis; Nerver growth factor; NF-Kappa B

Oncomodulin/truncated protamine-mediated Nogo-66 receptor small interference RNA delivery promotes axon regeneration in retinal ganglion cells.

The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ∼12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.

Preprodynorphin‐expressing neurons constitute a large subgroup of somatostatin‐expressing GABAergic interneurons in the mouse neocortex

Dynorphins, leumorphin, and neoendorphins are preprodynorphin (PPD)-derived peptides and ligands for κ-opioid receptors. Using an antibody to PPD C-terminal, we investigated the chemical and molecular characteristics of PPD-expressing neurons in mouse neocortex. PPD-immunopositive neuronal somata were distributed most frequently in layer 5 and less frequently in layers 2-4 and 6 throughout neocortical regions. Combined labeling of immunofluorescence and fluorescent mRNA signals revealed that almost all PPD-immunopositive neurons expressed glutamic acid decarboxylase but not vesicular glutamate transporter, indicating their γ-aminobutyric acid (GABA)ergic characteristics, and that PPD-immunopositive neurons accounted for 15% of GABAergic interneurons in the primary somatosensory area. As GABAergic interneurons were divided into several groups by specific markers, we further examined the chemical characteristics of PPD-expressing neurons by the double immunofluorescence labeling method. More than 95% of PPD-immunopositive neurons were also somatostatin (SOM)-immunopositive in the primary somatosensory, primary motor, orbitofrontal, and primary visual areas, but only 24% were SOM-immunopositive in the medial prefrontal cortex. In the primary somatosensory area, PPD-immunopositive neurons constituted 50%, 79%, 55%, and 17% of SOM-immunopositive neurons in layers 2-3, 4, 5, and 6, respectively. Although SOM-expressing neurons contained calretinin-, neuropeptide Y-, nitric oxide synthase-, and reelin-expressing neurons as subgroups, only reelin immunoreactivity was detected in many PPD-immunopositive neurons. These results indicate that PPD-expressing neurons constitute a large subgroup of SOM-expressing cortical interneurons, and the PPD/SOM-expressing GABAergic neurons might serve not only as inhibitory elements in the local cortical circuit, but also as modulators for cortical neurons expressing κ-opioid and/or SOM receptors.

Immunofluorescence Labeling of Pollen Tubes

Pollen tube is regarded as an excellent single-cell model system in plant cell studies. This protocol describes the use of a rapid and reliable immunofluorescence labeling method for studying in situ localization of proteins in pollen tubes. The whole experiment contains two major steps: pollen tube in vitro germination, and pollen tube fixation and immunolabeling. It takes about 2 days from pollen tube germination to immunofluorescence detection.

흰쥐의 삼차신경절에서 Transient receptor potential ankyrin 1 (TRPA1)의 발현 특성에 관한 연구

Transient receptor potential ankyrin 1 (TRPA1)은 <TEX>$17^{\circ}C$</TEX>보다 낮은 유해한 온도 및 자극적인 화합물에 의해 활성화되며 통각을 조절한다. 그러나 TRPA1에 의한 통각정보가 어떻게 처리되는지에 대한 정보는 많이 알려져 있지 않다. 본 연구에서는 흰쥐의 삼차신경절에서 TRPA1을 발현하는 신경세포의 특성을 규명하기 위해서, 면역형광기법을 사용하여 TRPA1을 발현하는 신경세포에서 다른 통각수용기들에서 발현되며, 특징적인 기능을 수행하는 수용기인 transient receptor potential vanilloid 1 (TRPV1)와 <TEX>$P2X_3$</TEX>와의 발현양상을 조사하였다. TRPA1을 발현하는 신경세포에서 열감각수용기이며, 통각표지자인 TRPV1과의 공존을 조사해 본 결과, TRPA1 면역양성 신경세포 중에서 58.8% (328/558)가 TRPV1을 동시에 발현하였으며, 41.2% (230/558)가 TRPA1만 발현하고 TRPV1을 발현하지 않았다. TRPA1을 발현하는 신경세포 중 TRPV1을 동시에 발현하는 신경세포는 대부분 작거나 중간크기였다. 또한 TRPA1과 조직의 손상, 그리고 염증 시 분비되는 ATP와 결합하는 <TEX>$P2X_3$</TEX>와의 공존을 조사해 본 결과, TRPA1 면역양성 신경세포 중에서 26.1% (310/1186)의 신경세포에서 <TEX>$P2X_3$</TEX>을 동시에 발현하였으며, 73.9% (876/1186)의 신경세포에서 TRPA1만 발현하였다. TRPA1을 발현하는 신경세포 중 <TEX>$P2X_3$</TEX>을 동시에 발현하는 신경세포는 대부분 작거나 중간크기였다. 이러한 결과는 TRPA1을 발현하는 신경세포가 TRPV1 또는 <TEX>$P2X_3$</TEX>를 동시에 발현함으로써 동일한 신경세포가 구강안면영역에서의 냉통각 및 열통각을 조절할 뿐 아니라, 냉통각 및 염증성동통을 동시에 전달하는 등 하나의 신경세포가 여러 가지 통각의 전달에 관여하는 것을 시사한다. Transient receptor potential ankyrin 1 (TRPA1), responding to noxious cold (<TEX>${\leq}17^{\circ}C$</TEX>) and pungent compounds, is implicated in nociception, but little is known about the coexpression of TRPA1 and other channels or receptors involved in the nociception in craniofacial regions. To address this issue, we characterized the TRPA1-immunopositive (+) neurons in the rat trigeminal ganglion (TG) and investigated their colocalization with other proteins known to be expressed in nociceptive neurons, such as transient receptor potential vanilloid (TRPV1) and <TEX>$P2X_3$</TEX> receptor, using light microscopic immunofluorescence labeling method with TRPA1 and TRPV1 or <TEX>$P2X_3$</TEX> antisera. The majority of TRPA1+ neurons costained for TRPV1 (TRPV1+/TRPA1+; 58.8%, 328/558) and 41.2% only expressed TRPA1 but not TRPV1. The TRPV1+/TRPA1+ neurons were small and medium sized. In addition, we investigated the colocalization of TRPA1 with <TEX>$P2X_3$</TEX>, a nonselective cation channel activated by ATP that may be released in the extracellular space as a result of tissue damage and inflammation. Among all TRPA1+ TG neurons, 26.1% (310/1186) costained for <TEX>$P2X_3$</TEX>, whereas 73.9% (876/1186) of TRPA1+ neurons did not coexpress <TEX>$P2X_3$</TEX>. <TEX>$P2X_3$</TEX>+/TRPA1+ neurons were predominantly small and medium sized. These results suggest that TRPA1+ neurons coexpressing TRPV1 or <TEX>$P2X_3$</TEX> are involved in specific roles in the transmission and processing of orofacial nociceptive information by noxious cold, heat, and inflammation.

In-solution Staining and Arraying Method for the Immunofluorescence Detection of γH2AX Foci Optimized for Clinical Applications

Immunofluorescence quantification of γH2AX foci is a powerful approach to quantify DNA double-strand breaks induced by cancer therapy or accidental exposure to ionizing radiation. Here we report a modification to the γH2AX immunofluorescence labeling method, whereby cells are stained in-solution before being spotted and fixed onto microscope slides. Our modified method allows arraying of 16 patient samples/slide ready for foci counting in 2 h and demonstrated reliably detection of γH2AX foci in mononuclear cells prepared from patients who had undergone radiation therapy.

A Role for Fibrillar Collagen Deposition and the Collagen Internalization Receptor Endo180 in Glioma Invasion

BackgroundGlioblastoma multiforme (GBM, WHO grade IV) is the most common and most malignant of astrocytic brain tumors, and is associated with rapid invasion into neighboring tissue. In other tumor types it is well established that such invasion involves a complex interaction between tumor cells and locally produced extracellular matrix. In GBMs, surprisingly little is known about the associated matrix components, in particular the fibrillar proteins such as collagens that are known to play a key role in the invasion of other tumor types.Methodology/Principal FindingsIn this study we have used both the Masson's trichrome staining and a high resolution multiple immunofluorescence labeling method to demonstrate that intratumoral fibrillar collagens are an integral part of the extracellular matrix in a subset of GBMs. Correlated with this collagen deposition we observed high level expression of the collagen-binding receptor Endo180 (CD280) in the tumor cells. Further, interrogation of multiple expression array datasets identified Endo180 as one of the most highly upregulated transcripts in grade IV GBMs compared to grade III gliomas. Using promoter analysis studies we show that this increased expression is, in part, mediated via TGF-β signaling. Functionally, we demonstrate that Endo180 serves as the major collagen internalization receptor in GBM cell lines and provide the first evidence that this activity is critical for the invasion of GBM cells through fibrillar collagen matrices.Conclusions/SignificanceThis study demonstrates, for the first time, that fibrillar collagens are extensively deposited in GBMs and that the collagen internalization receptor Endo180 is both highly expressed in these tumors and that it serves to mediate the invasion of tumor cells through collagen-containing matrices. Together these data provide important insights into the mechanism of GBM invasion and identify Endo180 as a potential target to limit matrix turnover by glioma cells and thereby restrict tumor progression.