Figure 1. GluN1 expression in cultured cortical neurons visualized using HB7535.
HB7535 stains GluN1 receptors in a cultured rat cortical neuron using HB6581 (chicken polyclonal anti-MAP2 antibody) counterstain. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Antigen retrieval was carried out by heating the coverslip to 95°C in 100mM Tris, 5% urea, pH9.5. Following washes, cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7535 (mouse monoclonal anti-GluN1) and HB6581 (chicken polyclonal anti-MAP2) antibodies were incubated overnight (4°C) at a 1:1,000 dilution. This was followed by a one-hour incubation with secondary antibodies (Polyclonal goat anti-chicken Alexa Fluor 488 conjugated, 1:300 dilution and polyclonal goat anti-mouse DyLight 550 conjugated, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (11.1% power, PMT: 716V gain), 488nm (1% power, Hyd: 10% gain) and 561nm (1% power, Hyd: 10% gain) laser lines in a z-stack (0.44µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. GluN1 histoblot in a horizontal rat brain section using HB7535.
HB7535 reveals the distribution of GluN1 expression within the rat brain with especially high expression seen in the molecular layer and stratum radiatum of the hippocampus. Abbreviations: Al: Insular cortex, Au: Auditory cortex, CA1: CA1 region of hippocampus, CA3: CA3 region of hippocampus, CB: Cerebellum, cc: Corpus callosum, Cg2: Cingulate cortex, Cpu: Caudate putamen, D3V: Dorsal third ventricle, DG: Dentate gyrus, DpG: Deep grey layer superior collic, Ent: Entorhinal cortex, Fra: Frontal association cortex, LTN: Lateral thalamic nuclei, M1: Primary motor cortex, OC: Orbital cortex, PrL: Prelimbic cortex, SS: Somatosensory cortex, TeA: Temporal association cortex. Method: Histoblots were performed following the methodology detailed in Molar, 2016. Neuromethods. In brief: 10µm fresh frozen sections were prepared on a cryostat from rat brains and transferred from slides to nitrocellulose membranes. Membranes were incubated in DNase I for 20 minutes at 37°C before incubation in stripping buffer containing β-mercaptoethanol for 1hr at 45°C. Sections were then blocked in 5% non-fat dry milk in TBST for 1hr before being incubated in HB7535 at 1µg/ml (1:1000 dilution) overnight at 4°C. Following washing, membranes were incubated in secondary antibody (goat anti-mouse alkaline phosphatase conjugated, Thermofisher A16069) at a 1:5,000 dilution for 90 minutes at room temperature. Membranes were developed using HB1881 NBT/BCIP solution before colour development was stopped with PBS. Membranes were imaged on a high-resolution desktop scanner.
Figure 3. GluN1 and NeuN expression in rat cerebellum mapped using HB7535.
HB7535 labels the dense expression of GluN1 receptors in the cerebellum. Method: Rat brains were dissected and fixed overnight in 9% glyoxal, 8% acetic acid, pH4 before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices. Sections were then blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB7535 (1:1000 dilution) and HB6498 (rabbit polyclonal anti-NeuN, 1:2,000 dilution, 0.5µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and goat anti-rabbit DyLight 650, Thermofisher 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured as a tilescan using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack using DAP, TX2 and Y5 filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. GluN1 expression in brain P2 membrane fractions when probed with HB7535.
HB7535 revealed a band of approximately 125kDa present almost exclusively in brain P2 fraction preparations (faint immunoreactivity is seen in mouse brain cytosol fraction which is likely due to the inability to exclude any membrane contamination during the subcellular fractionation process. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation) Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (NEB Prestained Protein standard) before being run at 60V for 30 minutes followed by 130V for 120 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7535 at a 1:1,000 dilution (1µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Following imaging, the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing, the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody (Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).
Figure 5. GluN1 expression in rat dentate gyrus mapped using HB7535.
HB7535 labels the dense expression of GluN1 receptors in the dentate gyrus and CA1. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices. Citrate antigen retrieval was carried out through incubation of sections with 10mM citric acid, 0.05% Tween 20, pH6 at 95°C for 30 minutes followed by a 20 minute cooling period. Sections were then blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB7535 (1:1000 dilution) and HB7266 (rabbit polyclonal anti-neurofilament L, 1:2,000 dilution, 0.5µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher, 35503 and goat anti-rabbit DyLight 594, Thermofisher 35561). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured as a tilescan using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 10x objective in a z-stack (5.4µm spacing). The image was captured using A4 (68ms exposure), GFP (376ms exposure) and Y3 (1s exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Colocalization of GluN1 and GluA1-4 in cultured cortical rat neurons using HB7535.
Both GluN1 and GluA1-4 are expressed in cultured rat cortical neurons and are visualised using HB7535 (mouse monoclonal anti-GluN1) and HB7534 (rabbit polyclonal anti-GluA1-4) antibodies using HB6581 (chicken polyclonal anti-MAP2 antibody) counterstain. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Antigen retrieval was carried out by heating the coverslip to 95°C in 100mM Tris, 5% urea, pH9.5. Following washes, cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7535 (mouse monoclonal anti-GluN1), HB7534 (rabbit polyclonal GluA1-4) and HB6581 (chicken polyclonal anti-MAP2) antibodies were incubated overnight (4°C) at a 1:1,000 dilution. This was followed by a one-hour incubation with secondary antibodies (Polyclonal goat anti-chicken Alexa Fluor 488 conjugated, 1:300 dilution, polyclonal goat anti-rabbit DyLight 650 conjugated, 1:300 dilution and polyoclonal goat anti-mouse DyLight 550 conjugated, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective (1.28x zoom), 405nm (11.1% power, PMT: 543V gain), 488nm (1% power, Hyd: 10% gain), 561nm (1% power, Hyd: 10% gain) and 633nm (5% power, Hyd: 18.6% gain) laser lines in a z-stack (0.38µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. GluN1 expression only observed within brain P2 membrane fractions when probed with HB7535.
HB7535 revealed a band of approximately 116kDa present only in brain P2 fraction preparations. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation) Samples were loaded (20µg / lane) onto a 7.5% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour) before being run at 60V for 30 minutes followed by 120V for 110 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7535 at a 1:1,000 dilution (1µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure).
Figure 8. GluN1 receptors on the dendrite of a cultured rat cortical neuron visualized using HB7535.
HB7535 staining of GluN1 receptors on the dendrite of a cultured rat cortical neuron using HB6581 (chicken polyclonal anti-MAP2 antibody) counterstain. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Antigen retrieval was carried out by heating the coverslip to 95°C in 100mM Tris, 5% urea, pH9.5. Following washes, cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7535 (mouse monoclonal anti-GluN1) and HB6581 (chicken polyclonal anti-MAP2) antibodies were incubated overnight (4°C) at a 1:1,000 dilution. This was followed by a one-hour incubation with secondary antibodies (Polyclonal goat anti-chicken Alexa Fluor 488 conjugated, 1:300 dilution and polyoclonal goat anti-mouse DyLight 550 conjugated, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective (3.89x zoom), 405nm (11.1% power, PMT: 673V gain), 488nm (1% power, Hyd: 10% gain) and 561nm (1% power, Hyd: 10% gain) laser lines in a z-stack (0.38µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 9. GluN1 histoblot in a horizontal rat brain section using HB7535.
HB7535 reveals the distribution of GluN1 expression within the rat brain with especially high expression seen in the hippocampus and cortex. Abbreviations: ON: Olfactory nucleus, Pir: Piriform cortex, IC: Insular cortex, PRh: Perirhinal cortex, CA1: CA1 region of hippocampus, CA3: CA3 region of hippocampus, DG: Dentate gyrus, Ent: Entorhinal cortex, Cpu: Caudate putamen, MS: Medial septum, GP: Globus pallidus, TN: Thalamic nuclei, Rt: Reticular formation, mlf: medial longitudinal fasciculus, CB: Cerebellum. Method: Histoblots were performed following the methodology detailed in Molar, 2016. Neuromethods. In brief: 10µm fresh frozen sections were prepared on a cryostat from rat brains and transferred from slides to nitrocellulose membranes. Membranes were incubated in DNase I for 20 minutes at 37°C before incubation in stripping buffer containing β-mercaptoethanol for 1hr at 45°C. Sections were then blocked in 5% non-fat dry milk in TBST for 1hr before being incubated in HB7535 at 1µg/ml (1:1000 dilution) overnight at 4°C. Following washing, membranes were incubated in secondary antibody (goat anti-mouse alkaline phosphatase conjugated, Thermofisher A16069) at a 1:5,000 dilution for 90 minutes at room temperature. Membranes were developed using HB1881 NBT/BCIP solution before colour development was stopped with PBS. Membranes were imaged on a high-resolution desktop scanner.
Figure 10. Concentration response of HB7535 staining in a rat brain P2 preparation.
HB7535 shows strong affinity for GluN1 with bands being visible at concentrations as low as ≈19ng/ml. Method: P2 membrane fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). P2 samples were loaded (20µg / lane) onto a 7.5% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour) before being run at 60V for 45 minutes followed by 130V for 80 minutes. Wet transfer to a PVDF membrane was completed in 120 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7535. Each strip was incubated separately with a separate HB7535 concentration with this ranging from 1200ng/ml (1:250 dilution) to 4.72ng/ml (1:64,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.
Figure 11. GluN1 histoblot in a horizontal rat brain section using HB7535.
HB7535 reveals the distribution of GluN1 expression within the rat brain with especially high expression seen in the hippocampus and cortex. Abbreviations: ON: Olfactory nucleus, Pir: Piriform cortex, IC: Insular cortex, PRh: Perirhinal cortex, CA1: CA1 region of hippocampus, CA3: CA3 region of hippocampus, DG: Dentate gyrus, Ent: Entorhinal cortex, Cpu: Caudate putamen, MS: Medial septum, GP: Globus pallidus, TN: Thalamic nuclei, Rt: Reticular formation, mlf: medial longitudinal fasciculus, CB: Cerebellum. Method: Histoblots were performed following the methodology detailed in Molar, 2016. Neuromethods. In brief: 10µm fresh frozen sections were prepared on a cryostat from rat brains and transferred from slides to nitrocellulose membranes. Membranes were incubated in DNase I for 20 minutes at 37°C before incubation in stripping buffer containing β-mercaptoethanol for 1hr at 45°C. Sections were then blocked in 5% non-fat dry milk in TBST for 1hr before being incubated in HB7535 at 1µg/ml (1:1000 dilution) overnight at 4°C. Following washing, membranes were incubated in secondary antibody (goat anti-mouse alkaline phosphatase conjugated, Thermofisher A16069) at a 1:5,000 dilution for 90 minutes at room temperature. Membranes were developed using HB1881 NBT/BCIP solution before colour development was stopped with PBS. Membranes were imaged on a high-resolution desktop scanner.
Figure 1. GluN1 expression in cultured cortical neurons visualized using HB7535.
HB7535 stains GluN1 receptors in a cultured rat cortical neuron using HB6581 (chicken polyclonal anti-MAP2 antibody) counterstain. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Antigen retrieval was carried out by heating the coverslip to 95°C in 100mM Tris, 5% urea, pH9.5. Following washes, cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7535 (mouse monoclonal anti-GluN1) and HB6581 (chicken polyclonal anti-MAP2) antibodies were incubated overnight (4°C) at a 1:1,000 dilution. This was followed by a one-hour incubation with secondary antibodies (Polyclonal goat anti-chicken Alexa Fluor 488 conjugated, 1:300 dilution and polyclonal goat anti-mouse DyLight 550 conjugated, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (11.1% power, PMT: 716V gain), 488nm (1% power, Hyd: 10% gain) and 561nm (1% power, Hyd: 10% gain) laser lines in a z-stack (0.44µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. GluN1 histoblot in a horizontal rat brain section using HB7535.
HB7535 reveals the distribution of GluN1 expression within the rat brain with especially high expression seen in the molecular layer and stratum radiatum of the hippocampus. Abbreviations: Al: Insular cortex, Au: Auditory cortex, CA1: CA1 region of hippocampus, CA3: CA3 region of hippocampus, CB: Cerebellum, cc: Corpus callosum, Cg2: Cingulate cortex, Cpu: Caudate putamen, D3V: Dorsal third ventricle, DG: Dentate gyrus, DpG: Deep grey layer superior collic, Ent: Entorhinal cortex, Fra: Frontal association cortex, LTN: Lateral thalamic nuclei, M1: Primary motor cortex, OC: Orbital cortex, PrL: Prelimbic cortex, SS: Somatosensory cortex, TeA: Temporal association cortex. Method: Histoblots were performed following the methodology detailed in Molar, 2016. Neuromethods. In brief: 10µm fresh frozen sections were prepared on a cryostat from rat brains and transferred from slides to nitrocellulose membranes. Membranes were incubated in DNase I for 20 minutes at 37°C before incubation in stripping buffer containing β-mercaptoethanol for 1hr at 45°C. Sections were then blocked in 5% non-fat dry milk in TBST for 1hr before being incubated in HB7535 at 1µg/ml (1:1000 dilution) overnight at 4°C. Following washing, membranes were incubated in secondary antibody (goat anti-mouse alkaline phosphatase conjugated, Thermofisher A16069) at a 1:5,000 dilution for 90 minutes at room temperature. Membranes were developed using HB1881 NBT/BCIP solution before colour development was stopped with PBS. Membranes were imaged on a high-resolution desktop scanner.
Figure 3. GluN1 and NeuN expression in rat cerebellum mapped using HB7535.
HB7535 labels the dense expression of GluN1 receptors in the cerebellum. Method: Rat brains were dissected and fixed overnight in 9% glyoxal, 8% acetic acid, pH4 before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices. Sections were then blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB7535 (1:1000 dilution) and HB6498 (rabbit polyclonal anti-NeuN, 1:2,000 dilution, 0.5µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and goat anti-rabbit DyLight 650, Thermofisher 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured as a tilescan using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack using DAP, TX2 and Y5 filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. GluN1 expression in brain P2 membrane fractions when probed with HB7535.
HB7535 revealed a band of approximately 125kDa present almost exclusively in brain P2 fraction preparations (faint immunoreactivity is seen in mouse brain cytosol fraction which is likely due to the inability to exclude any membrane contamination during the subcellular fractionation process. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation) Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (NEB Prestained Protein standard) before being run at 60V for 30 minutes followed by 130V for 120 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7535 at a 1:1,000 dilution (1µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Following imaging, the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing, the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody (Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).
Figure 5. GluN1 expression in rat dentate gyrus mapped using HB7535.
HB7535 labels the dense expression of GluN1 receptors in the dentate gyrus and CA1. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices. Citrate antigen retrieval was carried out through incubation of sections with 10mM citric acid, 0.05% Tween 20, pH6 at 95°C for 30 minutes followed by a 20 minute cooling period. Sections were then blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB7535 (1:1000 dilution) and HB7266 (rabbit polyclonal anti-neurofilament L, 1:2,000 dilution, 0.5µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 488, Thermofisher, 35503 and goat anti-rabbit DyLight 594, Thermofisher 35561). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured as a tilescan using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 10x objective in a z-stack (5.4µm spacing). The image was captured using A4 (68ms exposure), GFP (376ms exposure) and Y3 (1s exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Colocalization of GluN1 and GluA1-4 in cultured cortical rat neurons using HB7535.
Both GluN1 and GluA1-4 are expressed in cultured rat cortical neurons and are visualised using HB7535 (mouse monoclonal anti-GluN1) and HB7534 (rabbit polyclonal anti-GluA1-4) antibodies using HB6581 (chicken polyclonal anti-MAP2 antibody) counterstain. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Antigen retrieval was carried out by heating the coverslip to 95°C in 100mM Tris, 5% urea, pH9.5. Following washes, cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7535 (mouse monoclonal anti-GluN1), HB7534 (rabbit polyclonal GluA1-4) and HB6581 (chicken polyclonal anti-MAP2) antibodies were incubated overnight (4°C) at a 1:1,000 dilution. This was followed by a one-hour incubation with secondary antibodies (Polyclonal goat anti-chicken Alexa Fluor 488 conjugated, 1:300 dilution, polyclonal goat anti-rabbit DyLight 650 conjugated, 1:300 dilution and polyoclonal goat anti-mouse DyLight 550 conjugated, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective (1.28x zoom), 405nm (11.1% power, PMT: 543V gain), 488nm (1% power, Hyd: 10% gain), 561nm (1% power, Hyd: 10% gain) and 633nm (5% power, Hyd: 18.6% gain) laser lines in a z-stack (0.38µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. GluN1 expression only observed within brain P2 membrane fractions when probed with HB7535.
HB7535 revealed a band of approximately 116kDa present only in brain P2 fraction preparations. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation) Samples were loaded (20µg / lane) onto a 7.5% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour) before being run at 60V for 30 minutes followed by 120V for 110 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7535 at a 1:1,000 dilution (1µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure).
Figure 8. GluN1 receptors on the dendrite of a cultured rat cortical neuron visualized using HB7535.
HB7535 staining of GluN1 receptors on the dendrite of a cultured rat cortical neuron using HB6581 (chicken polyclonal anti-MAP2 antibody) counterstain. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Antigen retrieval was carried out by heating the coverslip to 95°C in 100mM Tris, 5% urea, pH9.5. Following washes, cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7535 (mouse monoclonal anti-GluN1) and HB6581 (chicken polyclonal anti-MAP2) antibodies were incubated overnight (4°C) at a 1:1,000 dilution. This was followed by a one-hour incubation with secondary antibodies (Polyclonal goat anti-chicken Alexa Fluor 488 conjugated, 1:300 dilution and polyoclonal goat anti-mouse DyLight 550 conjugated, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective (3.89x zoom), 405nm (11.1% power, PMT: 673V gain), 488nm (1% power, Hyd: 10% gain) and 561nm (1% power, Hyd: 10% gain) laser lines in a z-stack (0.38µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 9. GluN1 histoblot in a horizontal rat brain section using HB7535.
HB7535 reveals the distribution of GluN1 expression within the rat brain with especially high expression seen in the hippocampus and cortex. Abbreviations: ON: Olfactory nucleus, Pir: Piriform cortex, IC: Insular cortex, PRh: Perirhinal cortex, CA1: CA1 region of hippocampus, CA3: CA3 region of hippocampus, DG: Dentate gyrus, Ent: Entorhinal cortex, Cpu: Caudate putamen, MS: Medial septum, GP: Globus pallidus, TN: Thalamic nuclei, Rt: Reticular formation, mlf: medial longitudinal fasciculus, CB: Cerebellum. Method: Histoblots were performed following the methodology detailed in Molar, 2016. Neuromethods. In brief: 10µm fresh frozen sections were prepared on a cryostat from rat brains and transferred from slides to nitrocellulose membranes. Membranes were incubated in DNase I for 20 minutes at 37°C before incubation in stripping buffer containing β-mercaptoethanol for 1hr at 45°C. Sections were then blocked in 5% non-fat dry milk in TBST for 1hr before being incubated in HB7535 at 1µg/ml (1:1000 dilution) overnight at 4°C. Following washing, membranes were incubated in secondary antibody (goat anti-mouse alkaline phosphatase conjugated, Thermofisher A16069) at a 1:5,000 dilution for 90 minutes at room temperature. Membranes were developed using HB1881 NBT/BCIP solution before colour development was stopped with PBS. Membranes were imaged on a high-resolution desktop scanner.
Figure 10. Concentration response of HB7535 staining in a rat brain P2 preparation.
HB7535 shows strong affinity for GluN1 with bands being visible at concentrations as low as ≈19ng/ml. Method: P2 membrane fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). P2 samples were loaded (20µg / lane) onto a 7.5% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour) before being run at 60V for 45 minutes followed by 130V for 80 minutes. Wet transfer to a PVDF membrane was completed in 120 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7535. Each strip was incubated separately with a separate HB7535 concentration with this ranging from 1200ng/ml (1:250 dilution) to 4.72ng/ml (1:64,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.
Figure 11. GluN1 histoblot in a horizontal rat brain section using HB7535.
HB7535 reveals the distribution of GluN1 expression within the rat brain with especially high expression seen in the hippocampus and cortex. Abbreviations: ON: Olfactory nucleus, Pir: Piriform cortex, IC: Insular cortex, PRh: Perirhinal cortex, CA1: CA1 region of hippocampus, CA3: CA3 region of hippocampus, DG: Dentate gyrus, Ent: Entorhinal cortex, Cpu: Caudate putamen, MS: Medial septum, GP: Globus pallidus, TN: Thalamic nuclei, Rt: Reticular formation, mlf: medial longitudinal fasciculus, CB: Cerebellum. Method: Histoblots were performed following the methodology detailed in Molar, 2016. Neuromethods. In brief: 10µm fresh frozen sections were prepared on a cryostat from rat brains and transferred from slides to nitrocellulose membranes. Membranes were incubated in DNase I for 20 minutes at 37°C before incubation in stripping buffer containing β-mercaptoethanol for 1hr at 45°C. Sections were then blocked in 5% non-fat dry milk in TBST for 1hr before being incubated in HB7535 at 1µg/ml (1:1000 dilution) overnight at 4°C. Following washing, membranes were incubated in secondary antibody (goat anti-mouse alkaline phosphatase conjugated, Thermofisher A16069) at a 1:5,000 dilution for 90 minutes at room temperature. Membranes were developed using HB1881 NBT/BCIP solution before colour development was stopped with PBS. Membranes were imaged on a high-resolution desktop scanner.
Product information
Immunogen
Amino acids 1-564 of rat GluN1 expressed in a fusion protein
Epitope
Amino acids 341-561
Clone number
R1JHL
Isotype
IgG
Purification
Culture supernatent
Concentration
0.3mg/ml
Formulation
Lyophilised. When reconstituted contains PBS with 0.05% sodium azide and 1% recombinant albumin
Predicted species reactivity
Mouse, Rat, Human
Tested species reactivity
Mouse, Rat
Tested applications
Applications
WB, IHC(IF), Histoblot
Western blot optimal concentration
300ng/ml (1:1000 dilution) as tested in a rat brain P2 membrane preparation
IHC(IF) optimal concentration
300ng/ml (1:1000 dilution) as tested in rat brain hippocampal sections. Please note that utilisation of a citrate antigen retrieval protocol is required for successful staining.
ICC optimal concentration
300ng/ml (1:1000 dilution) as tested in cultured rat cortical neurons. Please note that utilisation of antigen retrieval is required for successful staining (10 minutes at 95°C in 100mM Tris, 5% urea, pH9.5).
Histoblot optimal concentration
300ng/ml (1:1000 dilution) as tested in horizontal rat brain sections
Product specific protocols
For IHC(IF) this antibody requires citrate antigen retrieval. For retrieval, incubate sections with 10mM citric acid, 0.05% Tween 20, pH6 at 95°C for 30 minutes followed by a 20 minute cooling period.
Positive control
GluN1 is widely expressed in the brain therefore neural tissues serve as an excellent positive control.
Negative control
Tissues such as the liver, heart and lung lack GluN1 expression while popular cell lines such as HeLa and HEK293 also lack expression therefore are good negative controls.
GluN1 has seven isoforms produced by alternative splicing:
Isoform 3 (canonical), known as Long isoform or NR1-3 - 938aa, 105.3kDa
Isoform 1, known as Short isoform or NR1-1 – 885aa, 99.3kDa
Isoform 2, known as Medium isoform or NR1-2 – 901aa, 101.2kDa
Isoform 4 – 922aa, 103.5kDa
Isoform 5 – 959aa, 107.9kDa
Isoform 6 – 943aa, 106.0kDa
Isoform 7 – 906aa, 101.9kDa
Expression
GluN1 is expressed alongside GluN2 as a heterotetramer in N-methyl-D-aspartate (NMDAR) receptors with each subunit containing two GluN1 subunits and a combination of two GluN2 (GluN2A, GluN2B, GluN2C or GluN2D) subunits. NMDAR receptors are expressed widely throughout the CNS and PNS. GluN1 expression has also been found in the kidney, heart and within bone alongside being reported in adipose tissue and the bladder.
Subcellular expression
GluN1 is expressed as part of NMDA receptors primarily within the post-synaptic densities found within the dendrites of neurones.
Processing
Following translation, the signal peptide (amino acids 1-18) is removed to leave the main peptide sequence.
Post translational modifications
GluN1 has multiple glycosylation sites alongside phosphorylation sites on residues 889, 890, 896 and 897.
Homology (compared to human)
Mouse GluN1 has a 99.04% homology to the human protein (9 amino acid changes) while Rat GluN1 has a 99.25% homology to human GluN1 (7 amino acid changes). Mouse and rat proteins have a 99.8% homology with only a single amino acid change (V460I).
Similar proteins
No other proteins with a significant homology were identified in a BLAST search. Other NMDAR subunits have <29% identity to GluN1.
Epitope homology (between species)
There is a 100% match between the epitope of HB7535 and human GluN1 while the mouse and rat proteins show 99.09% and 99.55% identity respectively.
Epitope homology (other proteins)
The only homology identified was with GluN3A and GluN3B however these were both of low similarity with identity scores of 33.1% and 33.2% respectively.
Storage & Handling
Storage instructions
-20°C then use reconstitution advice
Reconstitution advice
We recommend reconstituting with 50µl of either:
dH2O and storing at 4°C
50:50 ratio of dH2O to glycerol and storing at -20°C
dH2O then aliquot and store at -80°C
Take care when opening as the precipitate is extremely light and can easily be lost if disturbed. When reconstituting make sure that the antibody is thoroughly dissolved by pipetting up and down before giving the antibody a brief spin at <10,000g to make sure that all material is recovered and at the bottom of the tube.
Does this GluN1 antibody cross-react with other NMDA receptor subunits?
This clone has been reported to have no cross-reactivity with any GluN2 family subunits.
What guarantee do you have that my GluN1 antibody will perform as expected?
We guarantee that your GluN1 antibody will work for the applications and species we list on the datasheet. If the antibody fails to perform as expected then we are happy to offer a 100% refund guarantee. For more details please see our guarantee policy.
What protocols are available for use with this GluN1 antibody?
We have made a comprehensive collection of protocols that we have used in our experiments to validate this GluN1 antibody.
Will my GluN1 antibody work against species that have not been listed on the datasheet?
A species not being listed doesn’t mean that the GluN1 antibody won’t work, just that we haven’t tested it. If you test one of our antibodies in a new species please let us know (positive or negative)!
What counterstains do you recommend for use in ICC and IHC with this GluN1 antibody?
Western Blot Protocol (1 MB) 
