orai1  (Alomone Labs)

Journal: bioRxiv

Article Title: Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium

doi: 10.1101/2022.02.12.480222

Figure Lengend Snippet: Nrp2 expression in activated macrophages during joint inflammation A. Nrp2 expression on immune cells in CIA synovium. The hind paws of CIA-induced mice were digested and subjected to flow cytometric analysis. The representative histograms of Nrp2 staining and the cumulative data are shown. Data are expressed means ± SD. N = 3 from 3 independent experiments. B. The characteristics of Nrp2-positive macrophages. The representative histograms of CD86 and MHC class II expression on joint-infiltrating macrophages (left) and their cumulative data (right) are shown. N = 6 from 2 independent experiments. C. The expression of Nrp2 on BMMs. BMMs were cultured under indicated conditions, and Nrp2 expression was determined by flow cytometry. The representative histograms and the cumulative data are shown. N = 3 from 3 independent experiments. D. NRP2 expression in synovium-infiltrating cells in RA. The deposited single-cell RNA-seq data was re-analyzed, and several cell types were defined by UMAP. Expression of NRP2 is indicated with purple dots. The statistical analyses were performed using an unpaired t-test.

Article Snippet: Anti-Neuropilin2 (Nrp2) polyclonal antibody was from Alomone Labs.

Techniques: Expressing, Mouse Assay, Staining, Cell Culture, Flow Cytometry, RNA Sequencing Assay

Journal: Brain Structure & Function

Article Title: Store-operated calcium entry is essential for glial calcium signalling in CNS white matter

doi: 10.1007/s00429-017-1380-8

Figure Lengend Snippet: Mechanisms of SOCE in optic nerve glia. ATP-mediated calcium signalling in optic nerve glia is via P2Y G-protein-coupled receptors and the formation of IP3, which acts on IP3R1 on the ER to trigger release of Ca 2+ into the cytosol. Subsequent replenishment of ER stores in astrocytes and oligodendrocytes is dependent on SOCE via TRPM3 and Orai1, which form the plasmalemmal Ca 2+ channels, and mainly Stim1, which acts as the sensor of Ca 2+ depletion, and uptake into the ER is via SERCA pumps. Oligodendrocytes also express Stim2, which may be localized to the myelin, whereas Orai1, Stim1 and TRPM3 are localized to oligodendroglial somata. Notably, calcium homeostasis in optic nerve glia depends on an apparent continuous Ca 2+ influx from the extracellular milieu that is largely dependent on SOCE. Moreover, SOCE is essential for the sustainability of ATP-mediated Ca 2+ signalling in optic nerve glia, which has a central role in white matter physiology and pathology

Article Snippet: Primary antibodies were diluted in blocking solution and tissues/cells incubated overnight at 4 °C; anti-STIM1, anti-STIM2, anti-ORAI1, anti-TRPM3 were raised in rabbits (Alomone) and used at 1:300; chicken anti-GFAP (Chemicon) was used at 1:500.

Techniques:

Journal: Brain Structure & Function

Article Title: Store-operated calcium entry is essential for glial calcium signalling in CNS white matter

doi: 10.1007/s00429-017-1380-8

Figure Lengend Snippet: Expression of Orai/Stim transcripts in the mouse optic nerve. qRT-PCR was performed on lysates of acutely isolated optic nerves from WT postnatal mice (aged P9–P12) and adult mice (P30–P40). Data are from ten pooled optic nerves in each age group, run in triplicate, expressed as relative mRNA levels (2 -ΔCt ) compared to the housekeeping gene GAPDH (mean ± SEM, n = 3). Orai1 was the most highly expressed Orai isoform in the postnatal nerve (* p

Article Snippet: Primary antibodies were diluted in blocking solution and tissues/cells incubated overnight at 4 °C; anti-STIM1, anti-STIM2, anti-ORAI1, anti-TRPM3 were raised in rabbits (Alomone) and used at 1:300; chicken anti-GFAP (Chemicon) was used at 1:500.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay

Journal: Brain Structure & Function

Article Title: Store-operated calcium entry is essential for glial calcium signalling in CNS white matter

doi: 10.1007/s00429-017-1380-8

Figure Lengend Snippet: Expression of Orai/Stim in optic nerve oligodendrocytes. Immunolabelling for Orai1 ( a , green ), Stim1 ( b , green ) and Stim2 ( c , green ) in PLP-DsRed mice to identify oligodendrocytes ( a – c , red ), in optic nerve sections ( ai – iv, bi – iv, ci – iv ) and explant cultures ( av, bv, cv ). Confocal micrographs illustrate single channels ( ai, aii, bi, bii, ci, cii ) and merged cannels ( aiii, biii, ciii ). Expression of Orai1 and Stim1 is localized to oligodendroglial somata, whereas Stim2 immunostaining was primarily within the fascicles of myelinated axons, as demonstrated by the colocalization channels, illustrating voxels in which green and red channels are of equal intensity and appear yellow ( aiv, biv, civ ). Oligodendrocytes in explant cultures are immunopositive for Orai1 ( av ), Stim1 ( bv ) and Stim2 ( cv ). No immunostaining was observed in negative controls that were pre-incubated in blocking peptides for Orai1 ( inset , aiii ), Stim1 ( inset , biii ) and Stim2 ( inset , ciii ). Nuclei are stained with Hoechst blue. Scale bars a, b 10 µm, c 20 μm

Article Snippet: Primary antibodies were diluted in blocking solution and tissues/cells incubated overnight at 4 °C; anti-STIM1, anti-STIM2, anti-ORAI1, anti-TRPM3 were raised in rabbits (Alomone) and used at 1:300; chicken anti-GFAP (Chemicon) was used at 1:500.

Techniques: Expressing, Plasmid Purification, Mouse Assay, Immunostaining, Incubation, Blocking Assay, Staining

Journal: Brain Structure & Function

Article Title: Store-operated calcium entry is essential for glial calcium signalling in CNS white matter

doi: 10.1007/s00429-017-1380-8

Figure Lengend Snippet: Expression of Orai/Stim in optic nerve astrocytes. Double immunofluorescence labelling for Orai1 ( a , green ), Stim1 ( b , green ) and Stim2 ( c , green ), with GFAP to identify astrocytes ( a – c , red ), in WT optic nerve sections ( ai – iv , bi– iv, ci – iv ) and explant cultures ( av, bv, cv ). Confocal micrographs illustrate single channels ( ai, aii, bi, bii, ci, cii ) and merged cannels ( aiii, biii, ciii ). Expression of Orai1 and Stim1 is localized to astrocyte processes, whereas astrocytes were immunonegative for Stim2, as demonstrated by the colocalization channels, illustrating voxels in which green and red channels are of equal intensity and appear yellow ( aiv, biv, civ ). Astrocytes in explant cultures are immunopositive for Orai1 ( av ) and Stim1 ( bv ), but are immunonegative for Stim2 ( cv ). No immunostaining was observed in negative controls that were pre-incubated in blocking peptides for Orai1 ( inset , aiii ), Stim1 ( inset , biii ) and Stim2 ( inset , ciii ). Nuclei are stained with Hoechst blue. Scale bars a, b 10 µm, c 20 μm

Article Snippet: Primary antibodies were diluted in blocking solution and tissues/cells incubated overnight at 4 °C; anti-STIM1, anti-STIM2, anti-ORAI1, anti-TRPM3 were raised in rabbits (Alomone) and used at 1:300; chicken anti-GFAP (Chemicon) was used at 1:500.

Techniques: Expressing, Immunofluorescence, Immunostaining, Incubation, Blocking Assay, Staining

Journal: bioRxiv

Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

doi: 10.1101/2022.02.14.480443

Figure Lengend Snippet: Variable efficacy of Runx2-cre in deleting flox/flox Orai1 in osteoblasts. It is often assumed that promoter-cre constructs uniformly delete the target in all cells of an organ. Recent findings suggest that for reasons that are not clear this is sometimes not the case (see Text), we tested this by antibody labeling of Orai1 in wild type and Orai1fl/fl-Runx2cre bone. A . Rabbit antibody detects Orai1 in osteoblasts (upper left panel). Absence of antibody eliminates labeling (lower left panel). All sections are from one wild type animal. Left and right panels are of the same section. Osteoblasts are shown independently with phalloidin rhodamine (right panels). Fields are 200 µm across. B . In the wild type animal, osteoblasts are shown with the antibody at high power (fields 200 microns wide) and in phase of the same field (right). In the lower panels, the same field of a conditional KO animal. Some of the conditional KO cells (Orai1fl/fl-Runx2cre) do not label (arrows, phase and antibody label, lower panels). C . Lower power fields, 350 µm, of Orai1 labeled osteoblasts (surrounding tissue fluorescence is an artifact) in wild type bone (top) and conditional KO bone (bottom). Osteoblasts in the wild type label strongly, including surface cells of the bone (arrows, top). Some, but not all, of the osteoblasts in the conditional KO label (arrows, bottom). D . Western blot for Orai1 in cells expressing or not expressing the protein (See Fig 5 ). Thirty-five µg loads of cell protein from the isolates indicated were run on denaturing SDS-PAGE and blotted. This blot using the very specific Alomone antibody shows a trace of Orai1 even in the osteoblast KO preparation (left lane) and reduced, but not absent, Orai1 in the MSC KO (upper panel). The beta actin re-blot (lower panel) confirms similar protein loads.

Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

Techniques: Construct, Antibody Labeling, Labeling, Fluorescence, Western Blot, Expressing, SDS Page

Journal: bioRxiv

Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

doi: 10.1101/2022.02.14.480443

Figure Lengend Snippet: Key features of WT and conditional KO animals on static and dynamic histomorphometry. A . Bone volume/total volume (BV/TV) of vertebrae. Vertebral bone is significantly reduced (N=7, p=0.01) in the Orai1fl/fl-Runx2cre (Orai1 cKO) mice compared to controls. The greater variability in the conditional-knockout bone is thought to reflect variable cre excision; this variability is also apparent in the cortical section thickness ( Fig 2 ; see text). B . Trabecular thickness is significantly decreased in the Orai1 conditional knockout mice (N=7, p

Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

Techniques: Mouse Assay, Knock-Out

Journal: bioRxiv

Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

doi: 10.1101/2022.02.14.480443

Figure Lengend Snippet: Cross sections of vertebral cortex from wild-type (A) and Orai1 f/f -Runx2-cre conditional KO (B) animals. Images of H E stained histologic sections from upper lumbar vertebrae (top panels) are 1 mm wide; images from microCT scans of lower lumbar vertebrae (middle and lower panels) are 1.4 mm wide, with trabecular bone deleted. A . Wild type cortex with typical smooth bone showed relatively uniform thickness. B . The Orai1fl/fl-Runx2cre (abbreviated Orai1 cKO) animals had irregularly thinned regions (arrow) but no other distinguishing features. This is in keeping with the appearance of the bone surface in the three-dimensional reconstructions (see Fig 1). C . Cortical bone thickness, though variable, is reduced on average in Orai1fl/fl-Runx2cre (Orai1 cKO) animals (p = 0.009, N=8).

Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

Techniques: Staining

Journal: bioRxiv

Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

doi: 10.1101/2022.02.14.480443

Figure Lengend Snippet: Surface of wild type and Orai1fl/fl-Runx2cre fourth lumbar vertebrae. Bruker CTvox software-generated three-dimensional images of vertebrae reconstructed from microCT scans at 5 µm resolution. All animals were homozygous for floxed Orai1; the conditional knockouts (lower panels) are Runx2-cre positive. A . Representative vertebrae from control animals. Apart from sites of blood vessel entry, the surface of the bone is smooth, typical for mice at four months of age. B . Representative vertebrae from Orai1fl/fl-Runx2cre animals. In contrast to the control vertebrae, the bone surface appears irregular with patchy darker areas representing regions of reduced bone.

Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

Techniques: Software, Generated, Mouse Assay

Journal: bioRxiv

Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

doi: 10.1101/2022.02.14.480443

Figure Lengend Snippet: Elimination of Orai1 results in profoundly reduces OB differentiation and mineralization from OB precursors. Osteoblasts isolated as described in the methods section from control or Runx2-cre floxed (Orai1 f/f -Runx2-cre) conditional knock-out animals, were incubated 24 days in osteoblast mineralization medium and analyzed by histologic staining. Each well illustrated is 2 cm across. A . Von Kossa staining for mineral. Wild type cells made mineral nodules, but there were only rare and small nodules in Orai1 knockout cell cultures. Representative culture wells for control (WT) and conditional knockout (cKO) cells are shown on the left. Staining was quantified for four samples of each genotype. Mineralized matrix production appeared significantly reduced in cultures of Orai1-deficient osteoblasts (p

Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

Techniques: Isolation, Knock-Out, Incubation, Staining

Journal: The Journal of Neuroscience

Article Title: Glial Sulfatides and Neuronal Complex Gangliosides Are Functionally Interdependent in Maintaining Myelinating Axon Integrity

doi: 10.1523/JNEUROSCI.2095-18.2018

Figure Lengend Snippet: The additional loss of complex gangliosides on a sulfatide-null background does not augment disorganization at the NoR or electrophysiological function in the peripheral nervous system. A , There is modest Nav1.6 channel cluster domain lengthening in both CST −/− and CST −/− × GalNAc-T −/− teased SNs that does not reach significance. Representative images from teased SNs immunostained for Nav1.6 show that Nav channel cluster appearance was similar in every genotype. Disturbance of the paranode is indicated by invasion of Kv1.1 channels into the paranode, shown by a decrease in the gap between positive domains. The gap between Kv1.1-positive domains is significantly and comparably decreased in both CST −/− and CST −/− × GalNAc-T −/− mice compared with WT and GalNAc-T −/− NoRs. Representative images show that Kv1.1 formed two distinct domains of immunostaining at the juxtaparanodes in WT and GalNAcT −/− mice. Conversely, mice lacking sulfatide expression had Kv1.1 staining at the paranodes, suggesting disruption to the axo–glial junction. The length of pNFasc-immunostained domains does not significantly differ among genotypes. However, representative images show that labeling greatly differs: WT and GalNAc-T −/− NoRs have pNFasc staining that is most intense at the paranodes, forming two clear dimers (“normal”), whereas both CST −/− and CST −/− × GalNAc-T −/− mice have intense staining at the nodal gap (presumed NF186) with weaker paranodal (presumed NF155) staining (“abnormal”), which indicates a disturbance in paranodal adhesion (WT, n = 3; GalNAc-T −/− , n = 3; CST −/− , n = 3; and CST −/− × GalNAc-T −/− , n = 4). Scale bar, 5 μm. B , Perineural recordings of Nav and Kv channel currents from intercostal nerves showing an increase in recovery time for both peaks following paired pulse stimulation in both CST −/− and CST −/− × GalNAc-T −/− mice compared with WT and GalNAc-T −/− mice. The CST −/− and CST −/− × GalNAc-T −/− mice were not significantly different from each other at any ISI. Graphs display the means and ±SEM for each genotype (WT n = 5, GalNAc-T −/− n = 2, CST −/− n = 3, CST −/− × GalNAc-T −/− n = 2) and statistical analysis performed (two-way ANOVA; #WT vs CST −/− × GalNAc-T −/− mice; *WT vs CST −/− ; + GalNAc-T −/− vs CST −/− × GalNAc-T −/− ; ∧ GalNAc-T −/− vs CST −/− × GalNAc-T −/− ). SN conduction velocity decreased in CST −/− ( n = 4), GalNAc-T −/− ( n = 4), and CST −/− × GalNAc-T −/− ( n = 5) mice compared with WT ( n = 3), but only reached significance in CST −/− nerves. C , Peripheral nerve MAG immunostaining was comparable among genotypes. Scale bar, 20 μm. Graphs display the means and ±SEM for each genotype and statistical differences among genotypes were determined by one-way ANOVA followed by Tukey's post hoc tests to compare multiple comparisons, indicated on the graphs as follows: * p

Article Snippet: The following primary antibodies were used: mouse anti-phosphorylated neurofilament-H antibody (NF-H, BioLegend; RRID: AB_2715851 ; 1:2000); rat anti-MBP (Bio-Rad; RRID: AB_325004 ; 1:500); mouse anti-pan Nav (Sigma-Aldrich; RRID: AB_477552 ; 1:100); mouse anti-ankyrin G (Thermo Fisher Scientific; RRID: AB_2533145 ; 1:100); rabbit anti-Caspr (gifted from Professor Peles, Weizmann Institute, Israel; 1:1500); rabbit anti-Kv1.1 (Alomone Laboratories; RRID: AB_2040144 ; 1:200); rabbit anti-Nav1.6 (Sigma-Aldrich; RRID: AB_477480 ; 1:100); rabbit anti-pan neurofascin (anti-pNFasc; gifted from Professor Brophy, University of Edinburgh, UK; 1:1000); and mouse anti-MAG antibody (gifted from Professor Brophy, University of Edinburgh, UK; 1:100).

Techniques: Mouse Assay, Immunostaining, Expressing, Staining, Labeling