Four female young adult rhesus macaques (9-11 years) were perfused transcardially with artificial cerebrospinal fluid, followed by 4% paraformaldehyde, 0.05% glutaraldehyde and 0.18% picric acid in 0.1M phosphate buffer. The brains were vibrosliced coronally at 70μm, and sections of dlPFC were processed for peroxidase or gold immunocytochemistry. mGluR2/3 were labeled with rabbit antibodies against a 13-aa sequence of the shared C terminus of mGluR2 and mGluR3 (0.8μg/ml; ab6438, Abcam, Cambridge, MA), thus marking the cytoplasmic aspect of the plasma membrane (Figure 2 ) . Quantitative assessments were performed on peroxidase-labeled material using random, 21.6μm2 fields of the dlPFC neuropil (original magnification ×30,000), captured from the 4th surface-most ultrathin section of each plastic block; 5 fields/block, 5 blocks/brain. Detailed procedures have been described in 50 (link).
Ab6438
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab6438 is a mouse monoclonal antibody that recognizes the protein Tyrosine Kinase 2 (Tyk2). It is intended for use in immunohistochemistry applications.
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Lab products found in correlation
5 protocols using ab6438
1
Quantification of mGluR2/3 in Rhesus Macaque dlPFC
2
Quantitative Ultrastructural Analysis of mGluR2/3 in Rhesus Macaque dlPFC
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Four adult female rhesus macaques (9–11 years) were perfused transcardially with artificial cerebrospinal fluid, followed by 4% paraformaldehyde, 0.05% glutaraldehyde and 0.18% picric acid in 0.1m phosphate buffer. The brains were vibrosliced coronally at 70 μm, and sections of dlPFC were processed for peroxidase or gold immunocytochemistry. mGluR2/3 were labeled with rabbit antibodies against a 13-amino-acid sequence of the shared C terminus of mGluR2 and mGluR3 (0.8 μg ml−1; ab6438; Abcam, Cambridge, MA, USA), thus marking the cytoplasmic aspect of the plasma membrane (Figure 2 ). Quantitative assessments were performed on peroxidase-labeled material using random, 21.6 μm2 fields of the dlPFC neuropil (original magnification x30 000), captured from the 4th surface-most ultrathin section of each plastic block: 5 fields per block and 5 blocks per brain. Detailed procedures have been described in Paspalas et al.50 (link)
3
Visualizing Spinal Cord nNOS and mGluR2/3
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Mice were deeply anaesthetized with isoflurane, and perfused with a buffered 10% formaldehyde solution (Mildform 10N; FUJIFILM Wako). The spinal cords obtained from the lumbar enlargements were collected as soon as possible. The tissues were immersed in the same fixative at 4°C for 4 h or overnight, and equilibrated in 20% sucrose overnight at 4°C before cryoprotection. Sections with a thickness of 10 μm were prepared using a cryostat microtome (CM1520, Leica Microsystems), mounted on APS‐coated slide glasses (S8441; Matsunami, Osaka, Japan), and stored at –70°C until use. Sections were washed three times with 0.1 M PBS (pH 7.6) for 10 min, then treated with 2% goat serum for 60 min. The sections were primarily incubated with a goat anti‐nNOS antibody (1:100, ab1376; Abcam, Cambridge, UK) and a rabbit anti‐mGluR2 and anti‐mGluR3 antibody (1:100, ab6438; Abcam) for 2 days at 4°C. After washing, sections were secondarily incubated with a donkey anti‐goat IgG H&L conjugated with Alexa Fluor488 (to visualize nNOS; 1:1000, ab150129; Abcam), and a donkey anti‐rabbit IgG conjugated with rhodamine (to visualize mGluR2 and mGluR3; 1:2000 AP182R; EMD Millipore, Burtlington, MA, USA) overnight at 4°C. After washing, the sections were mounted using a medium containing DAPI (H‐1200; Vector Laboratories, Burlingame, CA, USA).
4
Characterization of mGluR Expression and Targeting in Prion Disease
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All compounds were purchased from Sigma-Aldrich unless otherwise stated. Monoclonal anti PrP antibody POM1 (1:5000) was generated as described previously [45 (link)]. Anti-mGluRs antibodies against representative receptors of each group, targeting the N-terminal domain were utilized: anti-mGluR5 #ab53090 (Abcam) or AB5675 (Millipore), anti-mGluR1 [EPR13540] (ab183712) (Abcam), anti-mGluR2+3 #ab6438 (Abcam) and anti-mGluR6 #AGC-026 (Alomone labs). Secondary antibodies were horseradish peroxidase (HRP)- conjugated rabbit anti–mouse IgG1 (1:10,000, Zymed) and goat anti–rabbit IgG1 (1:10,000, Zymed). Blots were developed using SuperSignal West Pico chemiluminescent substrate (Pierce) and visualized using the VersaDoc system (model 3000, Bio-Rad). Rocky Mountain Laboratory strain (RML; passage #6) prions (RML6) were amplified in CD1 mice by intracerebral inoculation into the lateral forebrain of 30 μl of 1% (wt/vol) brain homogenate. The mGluR5 antagonists MPEP and AFQ056 were kindly provided by Novartis. The mGluR1 antagonist YM202074 was purchased from Tocris Bioscience (Ellisville, USA).
5
Validating mGlu3 Receptor Antibody Specificity
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Omission of the primary antibody and substitution with non-immune rabbit serum abolished all reactivity. Peroxidase label was not detected when bridging secondary antibodies were excluded. Blocking of biotinylated probes with avidin/biotin also abolished the signal. Finally, additional electron microscopic immunocytochemistry performed in monkey dlPFC by our group showed that the antibody used in the present study exhibited a labeling pattern that was a subset of another commercially available and well-characterized antibody (Ab6438; Abcam) raised against the shared C-terminus of mGlu2 and mGlu3 (Jin et al., 2017 (link)), consistent with the labeling of mGlu3.
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