Anti darpp32

Manufactured by Merck Group

Sourced in United States

Anti-DARPP32 is a lab equipment product that functions as an antibody targeting the DARPP-32 protein. DARPP-32 is a protein involved in various cellular signaling pathways. The Anti-DARPP32 product can be used to detect and quantify the DARPP-32 protein in biological samples.

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DARPP32 (4 citations)

4 protocols using anti darpp32

Immunocytochemical Characterization of Differentiated Cells

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Cells were plated and fixed after 4 hours (as precursors) or 7 d (as differentiated cells), in 4% formaldehyde or 0.2% glutaraldehyde in 4% formaldehyde. Fluorescent immunocytochemistry was performed using standard protocols using the following primary antibodies: anti-β-Gal (1:1000, Promega), anti-Nestin (1:400, BD Pharm), anti-Oct4 (1:100, Santa Cruz), anti-Pax6 (1:50, DSHB), anti-Nkx2.1 (1:100, DAKO), anti-Mash1 (1:200, BDPharm), anti-β-III-tubulin (mouse 1:1000; rabbit 1:500, both Sigma), anti-GFAP (1:2000, DAKO), anti-GABA (1:500, Sigma), anti-DARPP32 (1:20,000, a gift from Paul Greengard) and anti-FoxP1 (1:500, Abcam). Secondary antibodies used were Alexa-fluor α 594 anti-mouse and α 488 anti-rabbit (both 1:200, DAKO).
Cells were visualised under UV fluorescence using a Leitz microscope and cell counts were performed using a grid randomly placed over 5 different fields per coverslip. The number of positive cells counted was expressed as mean ± SEM from replicates of 3–4 coverslips per condition. Images were processed using Optronics MagnaFire Software and Adobe Photoshop.

Precious S.V., Kelly C.M., Allen N.D, & Rosser A.E. (2016). Can manipulation of differentiation conditions eliminate proliferative cells from a population of ES cell-derived forebrain cells?. Neurogenesis, 3(1), e1127311.

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Quantitative Western Blot Analysis

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Western blot analysis was performed as previously described (Sarchielli et al., 2014) . Briefly, HSP cells were grown in F12 Coon's, treated with conditioned media and lysed in standard lysis buffer supplemented with protease inhibitor cocktail (Sigma Aldrich, Saint Louis, MO, USA). Aliquots containing 20 μg of proteins were loaded on SDS-PAGE, then transferred on poly-vinylidene difluoride membranes, blocked in 3% BSA and incubated with the following primary antibodies: anti-DARPP32 (1:1000, Sigma Aldrich) and anti-β actin (1:10000, Santa Cruz). The incubation with the primary antibodies was followed by peroxidase conjugated secondary IgG treatment and the reacted proteins were revealed by the enhanced chemiluminescence system (ECL extend, Euroclone, Milan, Italy). Image acquisition and densitometric analysis were performed with Quantity One software on a ChemiDoc XRS instrument (Bio-Rad Laboratories Inc.) and using β actin for normalization.

Idrizaj E., Sarchielli E., Morelli A., Garella R., Baccari M.C., Gallina P., Vannelli G.B., Francini F, & Squecco R. (2018). Cortical and spinal conditioned media modify the inward ion currents and excitability and promote differentiation of human striatal primordium. Journal of chemical neuroanatomy, 90.

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Immunohistochemical Staining Protocol

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The immunohistochemical procedure initiated with a 30 min dehydratation at 37°C followed by a 30 min hydration in 0.1 PBS and 1 h blocking in a 0.3% triton in 0.1 PBS with 10% normal goat serum both at room temperature (RT). The following primary antibodies diluted in a blocking solution with 0.1% triton were used: mouse monoclonal anti-ataxin-3 (1H9, Chemicon, Temecula, CA, USA; 1∶5000; 48 h, 4°C), rabbit polyclonal anti-β-galactosidase (Molecular Probes, Invitrogen; 1∶1000; 48 h, 4°C), rabbit polyclonal anti-DARPP-32 (Chemicon,Temecula, CA, USA; 1∶5000, 48 h, 4°C), rabbit polyclonal anti-calbindin D-28K (Chemicon,Temecula, CA, USA; 1∶1000, 48 h, 4°C). Sections were then incubated in secondary antibody, goat-anti rabbit and/or mouse conjugated to alexa 488 or 594 (Invitrogen) for 2 h/RT e and then mounted in mowiol (Sigma) with 4′,6′-diamidino-2-phenylindole.Fluorescence images were acquired with a Zeiss Axiovert 200 imaging microscope or LSM Zeiss microscope for double staining experiments.

Nóbrega C., Nascimento-Ferreira I., Onofre I., Albuquerque D., Déglon N, & Pereira de Almeida L. (2014). RNA Interference Mitigates Motor and Neuropathological Deficits in a Cerebellar Mouse Model of Machado-Joseph Disease. PLoS ONE, 9(8), e100086.

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Immunohistochemical Staining of Brain Samples

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Sections were first washed in PBS and then immersed in 0.3% H₂O₂ in PBS for 45 min to quench endogenous peroxidase activity. Following PBS washes, sections were immersed for 1 h in blocking solution [PBS containing 0.5% FBS, 0.3% Triton^TM X-100 and 1% bovine serum albumin (BSA)] and incubated overnight at 4°C with either rabbit anti-SREK1 (1:500, Abnova, H00140890-B01P), anti-TAF1 (1:1000, Sigma, HPA001075), anti-β-Gal (1:2000, Thermo Fisher, A-11132), anti-DARPP32 (1:5000, Chemicon, AB1656) or anti-cleaved caspase 3 (1:50, Cell Signaling, 9664) diluted in blocking solution. After washing, brain sections were incubated first with biotinylated goat anti-rabbit secondary antibody and then with avidin-biotin complex using the Elite Vectastain kit (Vector Laboratories, PK-6101-2). Chromogen reactions were performed with diaminobenzidine (SIGMAFAST™ DAB, Sigma, D4293) for 10 min. Mouse sections were mounted on glass slides and coverslipped with Mowiol^® (Calbiochem) while human sections where first dehydrated and then mounted with DePeX (SERVA). Images were captured using an Olympus BX41 microscope with an Olympus camera DP-70 (Olympus Denmark A/S).

Hernández I.H., Cabrera J.R., Santos-Galindo M., Sánchez-Martín M., Domínguez V., García-Escudero R., Pérez-Álvarez M.J., Pintado B, & Lucas J.J. (2020). Pathogenic SREK1 decrease in Huntington’s disease lowers TAF1 mimicking X-linked dystonia parkinsonism. Brain, 143(7), 2207-2219.

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