Seven- to eight- and 12–13-month-old mice were terminally anesthetized with isofluorane and perfused transcardially with heparinized saline (0.1%) followed by 4% paraformaldehyde (PFA) in phosphate buffer. Brains were removed and post-fixed in 4% PFA for 1 h before cryoprotection in 30% sucrose. Brains were frozen in isopentane chilled over dry ice and stored at −80 °C until use. Coronal sections were cut at 60 μm on a cryostat (CM1950, Leica), and stored in cryoprotection solution at −20 °C until use. For IHC, sections were rinsed in phosphate-buffered saline (PBS) and endogenous peroxidases were quenched in 1% hydrogen peroxide for 10 min. After rinses, sections were incubated in 2.5% normal donkey serum (Jackson labs) and MOM (Vector, for NeuN-staining) in 0.3% Triton X-100 in PBS. Sections were then incubated for 15–18 h at RT with either anti-NeuN (1:10,000; Millipore) or anti-DARPP-32 (1:300,000; Abcam). After washing, sections were incubated with a biotinylated secondary antibody (anti-mouse or rabbit, 1:500; Jackson ImmunoResearch). Staining of tissue-bound antibodies was visualized using a standard peroxidase-based method (Vectastain Elite, Vector Labs) with the 3,3-diaminobenzadine chromogen (DAB, Sigma, St. Louis, MO, USA).
Anti darpp32
Manufactured by Abcam
Anti-DARPP32 is a primary antibody that recognizes the DARPP-32 (dopamine and cAMP-regulated phosphoprotein) protein. DARPP-32 is a key regulator of dopamine and cAMP signaling pathways in the brain.
Automatically generated - may contain errors
Lab products found in correlation
Hdac1, by Abcam (2 mentions)
Anti atp5a, by Abcam (2 mentions)
Anti htt epr5526, by Abcam (2 mentions)
Anti polyq php3, by Abcam (2 mentions)
Anti pde10a, by Abcam (2 mentions)
Anti vinculin, by Merck Group (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti gfap, by Merck Group (2 mentions)
Anti polyq mw1, by Merck Group (2 mentions)
Anti htt mw8, by Developmental Studies Hybridoma Bank (2 mentions)
Anti neun, by Merck Group (2 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Cm1950, by Leica camera (1 mentions)
M o m, by Vector Laboratories (1 mentions)
Vectastain elite, by Vector Laboratories (1 mentions)
Glial fibrillary acidic protein (gfap), by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Odyssey system, by LI COR (1 mentions)
Dab staining, by Vector Laboratories (1 mentions)
Anti lamp1, by Abcam (1 mentions)
6 protocols using anti darpp32
1
Immunohistochemical Analysis of Neuronal Markers in Mouse Brains
2
Antibodies and Dilutions for Huntington's Disease
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The following antibodies and dilutions were used in this study: Anti-HTT Ab1 (aa1–17,1 (link)) 1:50 for capillary immunoassay and 1:2000 for western blot; anti-HTT EPR5526 (Abcam, Waltham, MA, ab109115, 1:2000 for western blot); anti-polyQ MW1 (MilliporeSigma, Burlington, MA, MABN2427, 1:50 for capillary immunoassay); anti-polyQ PHP3 (generous gift from Dr. Ali Khoshnan, 1:2000 for western blot); Anti-PDE10A (Abcam, Waltham, MA, #ab177933, 1:2000 for western blot); Anti-DARPP32 (Abcam, #ab40801, 1:2000 for capillary immunoassay); Anti-GFAP (MilliporeSigma, Burlington, MA, AB5804, 1:3000 for capillary immunoassay); Anti-GAPDH (MilliporeSigma, Burlington, MA, #MAB374, 1:10000 for western blot); Anti-Sodium channel subunit beta-4 (Abcam, Waltham, MA, #ab80539, 1:500 for western blot); Anti-vinculin (Sigma, St. Louis, MO, #V9131, 1:5000 for capillary immunoassay, 1:2000 for western blot); Anti-ATP5A (Abcam, Waltham, MA, #ab14748, 1:2000 for western blot); Anti-HTT MW8 (University of Iowa Developmental Studies Hybridoma Bank, 1:1000 for filter trap); Anti-HTT S830 (generous gift from Dr. Gillian Bates, 61 (link) 1:8000), HDAC1 (Abcam, Waltham, MA, ab32369–7, 1:4000).
3
Protein Extraction and Immunoblotting
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Whole cell protein extraction and Western blotting were performed as previously reported [20 (link)]. Membranes were probed with anti DARPP-32 (1:1000, Abcam), TH (1:2000, Sigma), GFAP (1:1000, Invitrogen) and anti beta-actin (1:2000, Millipore). Visualization and analysis of band intensities were performed using the Odyssey system (LICOR, Lincoln, NE, USA).
4
Immunostaining of Cell Lines and Brain Tissue
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A549 cells and brain sections were rinsed twice with PBST for 5 min at room temperature and then blocked with 0.5% bovine serum albumin in 1× PBST. After blocking, the samples were incubated overnight at 4 °C with anti-TH (1:1,000, Sigma), anti-DARPP32 (1:100, Abcam), anti-LC3 (1:100, Sigma), anti-p-α-synuclein (1:100, Abcam), anti-LAMP1 (1:100, Abcam), and anti-protein disulfide isomerase (PDI, 1:100, Invitrogen) primary antibodies. After overnight incubation, the samples were rinsed three times with 1× PBST (10 min per rinse). Immunofluorescence labeling was performed by incubating the cells and the brains with mouse anti-IgG Alexa Fluor (Invitrogen) and rabbit anti-IgG Alexa Fluor (Invitrogen). The cell nuclei were counterstained with DAPI (Invitrogen). The anti-DARPP32 and anti-TH antibodies were detected with 0.05% DAB staining (Vector Laboratories). The immunofluorescence was analyzed using confocal microscopy on a Zeiss LSM 780 confocal imaging system (Zeiss), whereas the DAB-stained samples were analyzed using bright-field microscopy.
5
Huntington's Disease Protein Immunoanalysis
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The following antibodies and dilutions were used in this study: Anti-HTT Ab1 (aa1–17,(Difiglia et al., 1995 (link))) 1:50 for capillary immunoassay and 1:2000 for western blot; anti-HTT EPR5526 (Abcam, Waltham, MA, ab109115, 1:2000 for western blot); anti-polyQ MW1 (MilliporeSigma, Burlington, MA, MABN2427, 1:50 for capillary immunoassay); anti-polyQ PHP3 (generous gift from Dr. Ali Khoshnan, 1:2000 for western blot); Anti-PDE10A (Abcam, Waltham, MA, #ab177933, 1:2000 for western blot); Anti-DARPP32 (Abcam, #ab40801, 1:2000 for capillary immunoassay); Anti-GFAP (MilliporeSigma, Burlington, MA, AB5804, 1:3000 for capillary immunoassay); Anti-GAPDH (MilliporeSigma, Burlington, MA, #MAB374, 1:10000 for western blot); Anti-Sodium channel subunit beta-4 (Abcam, Waltham, MA, #ab80539, 1:500 for western blot); Anti-vinculin (Sigma, St. Louis, MO, #V9131, 1:5000 for capillary immunoassay, 1:2000 for western blot); Anti-ATP5A (Abcam, Waltham, MA, #ab14748, 1:2000 for western blot); Anti-HTT MW8 (University of Iowa Developmental Studies Hybridoma Bank, 1:1000 for filter trap); Anti-HTT S830 (generous gift from Dr. Gillian Bates, (Landles et al., 2010 (link)) 1:8000), HDAC1 (Abcam, Waltham, MA, ab32369–7, 1:4000).
6
Immunohistochemical Staining of Rat Brain
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Rats were transcardially perfused and tissue was prepared, as previously described.44 (link) Sections were processed for Nissl staining using cresyl violet and immunohistochemistry with the following antibodies: anti-β-Gal (1:1000, Promega), anti-GFAP (1:2000, DAKO), anti-NeuN (1:4000, Chemicon), anti-doublecortin (DCX, 1:500, Abcam), anti-DARPP32 (1:10,000, a gift from Paul Greengard), anti-FoxP1 (1:500, Abcam), anti-calbindin (1:20,000, Sigma) and anti-parvalbumin (1:4000, Sigma). The basic protocol was the same for each antibody.
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