37-4600

antibody from Invitrogen Antibodies

Targeting: GJD2 CX36, GJA9

Provider product page for 37-4600

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [34]
Comments [0]
Validations
Immunohistochemistry [1]
Other assay [44]

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Product number: 37-4600 - Provider product page
Provider: Invitrogen Antibodies
Product name: Connexin 36 Monoclonal Antibody (1E5H5)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse
Host: Mouse
Isotype: IgG
Antibody clone number: 1E5H5
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

GJD2 protein structure - 37-4600 shown in red.

Supportive validation

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Experimental details: Figure 4 Newly generated anti-Cx30.2 antibodies (rabbit) are specific and reveal interaction of Cx30.2 and Cx36. (A) Western blot analysis of HeLa cell extracts transfected with different combinations of Cx30.2 and Cx36 and their enhanced green fluorescent protein (EGFP)-tagged variants. Blots were probed with anti-GFP, anti-Cx36 and newly generated anti-Cx30.2 antibodies from rabbit. Anti-Cx36 and anti-Cx30.2 antibodies recognize the respective connexin ( A,B , open arrowheads) and its fusion variant ( A,B , filled arrowheads). Please note that several bands become visible for untagged Cx30.2, pointing to different phosphorylation states (also see Supplementary Figure 1). These bands are not visible for the fusion protein. The Cx36 signal (lane 5) is extremely weak and nearly not visible but becomes apparent after immunoprecipitation (IP; B , lane 5). (B) IP using the same HeLa cell transfections as in (A) . Single or double transfections with untagged connexins did not show any bands in IP, as expected (lanes 1, 3, 9). Most importantly, when cells expressed Cx36-EGFP and untagged Cx30.2, Cx30.2 was detected in the IP (lane 7, lowest blot, open arrowhead); similarly, when cells expressed the Cx30.2-EGFP fusion protein together with Cx36, a band appeared for the native Cx36 (lane 8, middle blot, open arrowhead). These results suggest interaction of Cx30.2 and Cx36 in HeLa cells. Please note that the anti-Cx36 antibody produced several unspecific bands. The strong band at 25 k

Supportive validation

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Experimental details: Figure 3 Freeze-fracture replica immunogold labeling images of a spiny dendrite in stratum oriens , with gap junction double-labeled for Cx36 (6 and 18-nm gold beads ; within blue overlay ) and cluster of 10-nm E-face IMPs labeled for NR1 glutamate receptor subunits ( 12-nm gold beads within yellow overlays ) . (A) Low magnification stereoscopic images of one of three gap junctions labeled for Cx36 found in a 20 x 20 mum area of stratum oriens . A narrow branched spine [ arrow ; shown at higher magnification as (C) ] is consistent with identification of this neuronal plasma membrane E-face as either a portion of a CA3pyr dendrite (Chicurel and Harris, 1992 ) or of a spiny interneuron. A second branched spine is at the bottom ( red overlay ), separated from the co-planar dendritic shaft by ca. 20 nm, potentially indicating that the two membrane areas were continuous before fracturing. (B) Higher magnification stereoscopic image from (A) showing a medium-diameter gap junction ( blue overlay ) that is postsynaptic to a glutamatergic nerve terminal, identified by the presence of a shared PSD that is immunogold-labeled for NMDA-R1 by five 12-nm gold beads ( arrowheads ). Tilting, rotation, and stereoscopic analysis was required to clearly discern the PSD and gold labels beneath the almost vertical aspect of the replicated PSD in (A) . Note narrowing of the extracellular space ( black arrow ) at edge of gap junction. Air-drying of the high-relief replicas caused compression and art

Supportive validation

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Experimental details: Figure 6 Confocal maps showing exact locations of 20 Cx36-immunogold-labeled gap junctions found in adult rat hippocampus . (A,B) The labeled gap junctions shown in Figures 3 , 4 , 5 A-C, 11 , and 12 A,B are indicated by numbered yellow dots . Exact locations of seven other Cx36-labeled gap junctions ( unnumbered yellow dots ) found in these same two replicas are also indicated. In addition, the approximate locations of four other Cx36-labeled gap junctions found in three other replicas are indicated ( unnumbered blue dots ). Note the faint image of the gold ""index""TEM grid beneath and bonded to the tissue slice. Scale bars are 1 mm.

Supportive validation

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Experimental details: Figure 10 Dentate granule cell somatic and proximal dendrite plasma membrane E-face, with immunogold-labeled reticular gap junction . Large area of exposed neuronal plasma membrane ( red overlay ; ca. 1/4 of the 10 mum x 10 mum area is shown) is abutted by multiple large-diameter glutamatergic axon terminals ( purple overlays ). This replica was double-labeled for Cx35 + Cx36. Boxed areas are enlarged as stereoscopic images. (B) At intermediate magnification, active zones (*) are visible in the axon terminal P-face, and clusters of 10-nm E-face IMPs are seen in the subjacent granule cell proximal dendrite E-face, demonstrating two primary components defining multiple chemical synapses. In the center of the image is a two-lobed ""mushroom"" spine that, fortuitously, was shadowed deeply [enlarged as (C) ]. Several active zones (* and purple overlays ), one with a closely-associated PSD ( yellow overlay ), are indicated. A single gold bead above the replica, and therefore representing ""definitive noise,"" is indicated by "" ."" (C) At high-magnification, a medium-size (>150 connexons) ""reticular"" gap junction ( red overlay ) was faintly resolved deep in the spine head, but was easily detected because it is double-labeled by three 18-nm gold beads attached to rabbit polyclonal antibody for Cx35 (fish ortholog of Cx36; O'Brien et al., 2004 , 2006 ) and by 18 6-nm gold beads ( white arrows ) that label mouse monoclonal antibody for Cx36. Glutamate-receptor-like E-face IMPs

Supportive validation

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Experimental details: Figure 11 Images of freeze-fracture replica of immunogold labeled dendritic gap junctions in hippocampus . (A) Large reticular gap junction found between dendritic shafts of two probable interneurons in stratum oriens . It is labeled for Cx36 by ca. 88 6-nm gold beads and 26 18-nm gold beads. (B) Image of three Cx36-immunogold-labeled gap junctions between unidentified neuronal processes (axon terminals or dendrites). A nearby GABAergic synapse ( green overlay ) is on an unidentified cell process. The adjacent neuronal P-face ( purple overlay ) has the high density of IMPs associated with MF axon varicosities. Green overlay = GABA receptor 9-nm P-face IMPs. One of the gap junctions ( box ) is enlarged as an inset ( bottom center ). AZ, active zone of putative GABAergic synapse. Scale bars are 0.1 mum, unless otherwise indicated.

Supportive validation

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Experimental details: Figure 7 Stereoscopic images of large plaque and reticular gap junctions in adult male Mosquitofish. (A) Large plaque gap junction ( red overlay ) on a neuronal postsynaptic E-face labeled with antibodies against mouse/human Cx36 (36-4600, 6-nm [ white arrowheads ] and much more electron-dense 18-nm gold beads) and for GluR2 AMPA receptors (12-nm gold beads, white arrow ) ( Yellow arrow indicates either a 12-nm gold bead as ""noise"" on the Cx36-labeled gap junction or an anomalously small ""18-nm"" gold bead for Cx36). (B) High-magnification stereoscopic image of E-face of a neuronal reticular gap junction ( red overlay ) labeled for Cx35 (P-Ser276) by 6-nm ( white arrowheads ) and 18-nm gold beads. The black arrow points to ""cryptic"" labeling of connexins in an extended portion of the gap junction beneath the cross-fractured neuronal cytoplasm. Immediately adjacent to the gap junction is a cluster of E-face IMPs ( yellow overlay , not labeled) similar to other immunogold-labeled glutamate receptor channels. (C) P-face image showing three of more than a dozen unlabeled postsynaptic hemiplaques ( red overlays ) in the same replica as Figures 4 , 5B , where presynaptic hemiplaques are heavily labeled for Cx35 (P-Ser276). This absence of labeling in C is consistent with our demonstration in goldfish giant club ending/Mauthner cell mixed synapses that Cx35 is exclusively presynaptic and that Cx34.7 is exclusively postsynaptic (Rash et al., 2013 ). The postsynaptic P-face revea

Supportive validation

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Experimental details: Figure 1. Immunofluorescence staining of retinas showed selective disruption of Cx36 expression in cone photoreceptors in HRGP cre Gjd2 fl/fl . ( A, B ) Cx36 immunoreactive plaques were observed in the IPL and OPL of Gjd2 fl/fl and HRGP cre Gjd2 fl/fl mice (magnification: left 60x, right 240x); see black and white images in Supplementary Figure S1 . ( C ) Quantification of Cx36 immunoreactive plaques in retinas of HRGP cre Gjd2 fl/fl mice was lower compared to the littermate Gjd2 fl/fl control mice (*** P < 0.001, sample size: 6-7 mice / genotype). ( D ) Fluorescence was observed almost exclusively in cone photoreceptors in Ai6 (RCL-ZsGreen) Cre reporter mice. Retinal layers: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Supportive validation

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Experimental details: Figure 8 Cx36 expression in OFF alpha-RGCs in PV- Pten +/+ and PV- Pten +/- retinas. (A) A maximum projection of a Neurobiotin-filled OFF alpha-RGC (green) stained with Cx36 antibodies (red) in a PV- Pten +/+ retina at 1.0 mm from the optic nerve head. (B) A single optical section of the Neurobiotin-filled OFF alpha-RGC (green) stained with Cx36 antibodies (red) in PV- Pten +/+ retina. (C) Imaris reconstruction of panel (B) . Cx36 puncta closer than 0.3 mum to the dendrites are represented in red. (D) A maximum projection of a Neurobiotin-filled OFF alpha-RGC (green) stained with Cx36 antibodies (red) in PV- Pten +/- retina at 1.0 mm from the optic nerve head. (E) A single optical section of the Neurobiotin-filled OFF alpha-RGC (green) stained with Cx36 antibodies (red) in PV- Pten +/- retina. (F) Imaris reconstruction of panel (E) . Cx36 puncta closer than 0.3 mum to the dendrites are represented in red. (G) Histogram indicating the mean (+- SEM) of Cx36 puncta in OFF alpha-RGCs ( n = 3 cells from PV- Pten +/+ retinas, n = 5 cells from PV- Pten +/- retinas, * P < 0.05, Mann-Whitney test). Scale bar in (A,D) = 50 mum. Scale bar in (B,C,E,F) = 2 mum.

Supportive validation

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Experimental details: Figure 5. ipRGC-amacrine coupling is mediated in part by Cx36. ( A ) Cx36 immunostaining confirms nonglobal Cx36 knockout in Opn4 Cre/+ ; Cx36 flox/ flox ; Z/EG retinas. A1 : Cx36 immunostaining was imaged confocally in whole-mount retinas, and the z-stacks were rotated 90deg to show these orthogonal views of Opn4 Cre/+ ; Cx36 +/+ ; Z/EG ( left ) and Opn4 Cre/+ ; Cx36 flox/ flox ; Z/EG ( right ) retinas. A2 : Representative whole-mount images at focal planes within the outer ( top ) and inner ( bottom ) plexiform layers. ( B ) Neurobiotin staining patterns of six representative Opn4 Cre/+ ; Cx36 flox/ flox ; Z/EG ipRGCs. Arrowheads mark Neurobiotin-filled somas within the M3 ipRGC's dendritic field. ( C ) Population-averaged numbers of somas coupled to each Opn4 Cre/+ ; Cx36 flox/ flox ; Z/EG ipRGC of every type, including uncoupled ipRGCs ( black columns ). The number above each column is the number of ipRGCs analyzed for that ipRGC type. The Opn4 Cre/+ ; Z/EG control data ( gray columns ) have been replotted from Figure 1 C. *** P < 0.001.