Ab40801

Manufactured by Abcam

Sourced in United States

Ab40801 is a recombinant monoclonal antibody. It is designed for use in various immunoassay applications.

Automatically generated - may contain errors

Lab products found in correlation

Ab18465, by Abcam (2 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Mab5406, by Merck Group (1 mentions) Confocal microscopy, by Olympus (1 mentions) Gb23303, by Wuhan Servicebio Technology (1 mentions) Image pro plus 6.0 image analysis software, by Media Cybernetics (1 mentions) Gb2301, by Wuhan Servicebio Technology (1 mentions) Ab218532, by Abcam (1 mentions) Ab22625, by Abcam (1 mentions) Ab85367, by Abcam (1 mentions) Ab81296, by Abcam (1 mentions) T1299, by Merck Group (1 mentions) Dopamine elisa kit, by Abnova (1 mentions) Bca protein assay kit, by Solarbio (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Ab179471, by Abcam (1 mentions) Ab16502, by Abcam (1 mentions) Ab86299, by Abcam (1 mentions) Eclipse ti2, by Nikon (1 mentions) Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Ab40801 (EP720Y) (2 citations)

14 protocols using ab40801

Visualizing Microglial and DARPP32 Responses

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To examine the microglial response, cells were visualized using immunohistochemistry for IBA1, a microglia-specific protein up-regulated during activation[32 (link),33 (link)]. All incubations were done at room temperature unless otherwise noted. Sections were incubated in blocking solution (5% normal goat serum and 1% bovine serum albumin) with 0.2% Triton^TM-X-100 and 0.03% hydrogen peroxide in PBS for one hour. Anti-IBA1 (Wako 019-19741; 1:1,000 dilution) was bound to sections overnight at 4°C. Sections were next incubated in biotinylated secondary antibody (Vector Laboratories BA-1000; 1:200 dilution) for 10 minutes, followed by use of the Vectastain ABC Kit (Vector). 3,3'-diaminobenzidine (DAB) with the Metal Enhanced DAB Substrate Kit (Pierce) stained the reaction for visualization.
To visualize DARPP32-reactive cells, brain sections were incubated for 3 minutes in 3% hydrogen peroxide, 20 minutes in 0.2% Triton^TM-X-100, and 4 hours in 1.5% normal goat serum diluted in PBS. Anti-DARPP32 (Abcam ab40801; 1:10,000 dilution) in 1.5% normal goat serum bound sections at 4°C overnight. Biotinylated anti-rabbit antibody, ABC kit, and DAB reagent stained the reaction for visualization as above.

Johnson E., Chase K., McGowan S., Mondo E., Pfister E., Mick E., Friedline R.H., Kim J.K., Sapp E., DiFiglia M, & Aronin N. (2015). Safety of striatal infusion of siRNA in a transgenic Huntington's disease mouse model. Journal of Huntington's disease, 4(3), 219-229.

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Immunocytochemical Analysis of Neuronal Markers

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Cells cultured on coverslips were washed in PBS (pH 7.4), fixed in 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100. After incubation with 2% normal goat serum, fixed cells were incubated overnight at 4 °C with the following primary antibodies: anti-Tau (T1308-1, rPeptide, Athens, Georgia, USA 1:200), anti-DARPP-32 (ab40801, Abcam, 1:500), anti-MAP2 (4542, Cell Signaling, 1:500), anti-GAD67 (MAB5406, MilliporeSigma, 1:300), anti-Tubulin β 3 (Tuj1) (801201, BioLegend, San Diego, CA, USA, 1:500), and anti-Tom20 (11802-1-AP, Proteintech, 1:1000). Cells were washed in PBS (pH 7.4) and incubated with Alexa Fluor goat anti-mouse/rabbit 568 or 488 secondary antibodies (ThermoFisher Scientific, 1:1000), followed by incubation with Hoechst dye (1:10,000). Coverslips were mounted and slides were imaged by confocal microscopy (Olympus). MAP2⁺ neurite length, Tau⁺/Tuj1⁺ axonal length, and mitochondrial morphology were quantified using the ImageJ software.

Hu D., Sun X., Magpusao A., Fedorov Y., Thompson M., Wang B., Lundberg K., Adams D.J, & Qi X. (2021). Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington’s disease. Nature Communications, 12, 5305.

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Immunohistochemical Analysis of DARPP-32 and STAT3

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Stomach tissue sections were fixed in 4% paraformaldehyde at room temperature, embedded in paraffin, cut into 4 µm slices and mounted on glass slides. The sections were heated to 60°C for 20 min, de-paraffinized in xylene and dehydrated in alcohol. Antigen retrieval was performed in a pressure cooker using sodium citrate buffer (pH 6.0). The sections were then soaked in 3% H₂O₂/methanol solution to inhibit endogenous peroxidase activity, and then blocked with 3% bovine serum albumin (BSA). The sections were incubated overnight at 4°C with anti-DARPP-32 (1:50; ab40801, Abcam) and anti-STAT3 (1:300; #9139, Cell Signaling Technology, Inc.) primary antibodies. After washing for 10 min in phosphate buffered saline (PBS, pH 7.4), the sections were incubated for 20 min in horseradish peroxidase (HRP)-labeled secondary antibody, goat anti-rabbit or goat anti-mouse IgG (1:200; GB2301 and GB23303, Wuhan Servicebio Technology Co., Ltd.), and for 1 min in DAB reagent. The sections were then counterstained with hematoxylin prior to dehydration with ethanol and xylene. Images were obtained using a light microscope at 100×–200× magnification (Carl Zeiss AG), and the immunoreactivities of DARPP-32 and STAT3 were determined using Image Pro Plus 6.0 image analysis software (Media Cybernetics, Inc.).

Li D., Zhao L., Li Y., Kang X, & Zhang S. (2020). Gastro-Protective Effects of Calycosin Against Precancerous Lesions of Gastric Carcinoma in Rats. Drug Design, Development and Therapy, 14, 2207-2219.

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Dopamine and GLP-1 Signaling Pathways

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Salts and organic solvents used in solution preparations were purchased from Fisher Scientific (Leicestershire, UK), Sigma Chemicals (St. Louis, MO, USA, EUA), or Merck (Darmstad, Germany), with the highest grade of purity commercially available. It used antibodies to analyze D1R, D2R, DARPP32 (ab81296, ab85367, and ab40801 respectively, Abcam, UK), and tyrosine hydroxylase (TH), (T1299, Sigma Aldrich, St. Louis, MO, USA) at a 1:1000 dilution. Moreover, antibodies against GLP-1 and GLP-1R and against the phosphorylated form of insulin receptor were used (ab22625, ab218532, and InsR-Tyr972, ab5678 respectively, Abcam, UK) as well, against phosphorylated AMPK form (Thr172, 2535S, Cell Signalling Technology, Danvers, MA, USA, EUA). Calnexin was used as loading control (AB0037, Sicgen, Cantanhede, Portugal). Plasma dopamine levels and plasma GLP-1 levels were assessed through the Dopamine ELISA Kit, (Abnova, Taiwan) and Rat GLP1/Glucagon-like Peptide 1 ELISA Kit, (LifeSpan BioScience, Inc., Washington, DC, USA) respectively.

Tavares G., Rosendo-Silva D., Simões F., Eickhoff H., Marques D., Sacramento J.F., Capucho A.M., Seiça R., Conde S.V, & Matafome P. (2023). Circulating Dopamine Is Regulated by Dietary Glucose and Controls Glucagon-like 1 Peptide Action in White Adipose Tissue. International Journal of Molecular Sciences, 24(3), 2464.

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Quantification of Stomach Protein Levels

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Stomach tissues were ground in liquid nitrogen, homogenized on ice using lysis buffer containing protease inhibitors, and centrifuged at 12,000 rpm for 15 min to obtain the supernatant. Protein concentrations were quantified using a BCA protein assay kit (Solarbio Science & Technology Co., Ltd., Beijing, China). Equal amounts of proteins were loaded onto the SDS-PAGE gel electrophoresis system and the separated bands were transferred onto PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% nonfat milk for 1 h at room temperature, and then incubated at 4°C overnight with anti-integrin β1 (1:2000; ab179471, Abcam), anti-NF-κB (1:2000; ab16502, Abcam), anti-p-NF-κB (1:2000; ab86299, Abcam), anti-DARPP-32 (1:1000; ab40801, Abcam), and anti-STAT3 (1:1000; #9139, Cell Signaling Technology, Inc.) primary antibodies. Electrophoresis of anti-DARPP-32 was conducted with 12% gel, while 10% gel was used for the other antibodies. Subsequently, the membranes were washed and incubated with secondary antibodies for 1 h at room temperature. Routinely, protein load was monitored by using a super enhanced chemical luminescence reagent (ECL; Millipore, Bedford, MA, USA). The polypeptide bands from Western blotting were quantified by Image-J 1.46r software (National Institutes of Health, Bethesda, MD, USA), and normalized to GAPDH or actin (loading control).

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Immunohistochemical Analysis of Parkinson's and MSA

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Free-floating striatal and nigral sections of control (n = 3) and MSA (n = 3) brain tissue were first rinsed of cryoprotectant solution and then underwent citric acid heat mediated antigen retrieval. Nonspecific background staining was blocked by a 1-h incubation in a solution containing 2% bovine serum albumin and 3% of donkey serum. Tissue sections were incubated at room temperature overnight in the following primary antibodies: rabbit anti-Human CD3 (polyclonal, Dako A0452), mouse anti-HLA-DR (clone LN3, Invitrogen MA5-11966), rabbit anti-alpha-synuclein (phosphoS129) [EP1536Y] (polyclonal, abcam ab51253), mouse anti-alpha-synuclein (phosphoS129) [P-syn/81A] (monoclonal, abcam ab184674), rabbit anti-DARPP-32 [EP720Y] (polyclonal, abcam ab40801). Sections were washed of primary antibody, then incubated with appropriate Alexa-conjugated secondary antibodies (Life Technologies) at room temperature for 1 h. Sections were mounted on gelatin-coated slides, dehydrated through graded alcohol, cleared in xylene, and coverslipped with DPX mounting medium (Sigma-Aldrich). All images were obtained on a Nikon Eclipse Ti2 confocal microscope using a Nikon A1RHD camera. All figures were prepared using Photoshop 8.0 graphics software. Only minor adjustments of brightness and/or contrast were made.

Williams G.P., Marmion D.J., Schonhoff A.M., Jurkuvenaite A., Won W.J., Standaert D.G., Kordower J.H, & Harms A.S. (2020). T cell infiltration in both human multiple system atrophy and a novel mouse model of the disease. Acta Neuropathologica, 139(5), 855-874.

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Immunofluorescence Protocol for Neuronal Markers

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Cells were fixed with paraformaldehyde (4% in PBS) for 20 min, followed by permeabilization (0.2% Triton X-100 in PBS for 10 min) and blocking (3% BSA in 0.2% Triton X-100 in PBS for 10 min). Cells were incubated in primary antibody overnight at 4 °C (Mouse anti-Polyglutamine-Expansion Diseases Marker Antibody (Merck Millipore, MAB1574, 1:50), Rabbit anti-Cleaved Caspase-3 (Cell Signaling, 9661, 1:400), Rabbit anti-DARPP32 (Abcam, ab40801, 1:100), Chicken anti-MAP2 (Abcam, ab5392, 1:1,000). After washing with PBS, cells were incubated in secondary antibody (Alexa Fluor 488 goat anti-mouse (ThermoFisher Scientific, A-11029, 1:1,000), Alexa Fluor 647 donkey anti-chicken (Jackson ImmunoResearch, 703-605-155, 1:1,000), Alexa Fluor 568 goat anti-Rabbit (ThermoFisher Scientific A-21067, 1:1,000)) and co-stained with 2 μg ml⁻¹ Hoechst 33342 (Life technologies). Finally, coverslips were covered with Mowiol (Sigma).

Noormohammadi A., Khodakarami A., Gutierrez-Garcia R., Lee H.J., Koyuncu S., König T., Schindler C., Saez I., Fatima A., Dieterich C, & Vilchez D. (2016). Somatic increase of CCT8 mimics proteostasis of human pluripotent stem cells and extends C. elegans lifespan. Nature Communications, 7, 13649.

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Striatal Pathway Changes via IF

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Striatal changes of the direct and indirect pathways were investigated via immunofluorescence (IF) as previously described [23 (link)]. In brief, striatum slices (about 20~30 μm) were dehydrated with gradient alcohol. the following primary antibodies were applied: rabbit monoclonal anti-DARPP32 (1:100, ab40801, Abcam, MA, USA), rabbit polyclonal anti-Enkephalin (5 μg/ml, ab85798, Abcam). Images were obtained with a confocal laser scanning microscope (LSM700, Carl Zeiss, Germany) and analyzed via ImageJ software.

Cai H., Ni L., Hu X, & Ding X. (2021). Inhibition of endoplasmic reticulum stress reverses synaptic plasticity deficits in striatum of DYT1 dystonia mice. Aging (Albany NY), 13(16), 20319-20334.

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Co-Localization of Neuronal Markers

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In Figures 7, 8, DIG-labeled Drd1 and FLU-labeled Penk probes were labeled by Cy3 and Cy5 fluorescence signals as described above, respectively. To detect DARPP-32 immunoreactivity, the sections were then incubated with an anti-DARPP-32 antibody (1:500, Abcam ab40801) overnight at 4°C, followed by TNT wash and secondary antibody incubation (Alexa 488 conjugated-donkey anti-rabbit IgG antibody. 1:500; Jackson Immunoresearch Laboratories Inc. 711-545-152) for 1 h at room temperature. The sections were counterstained with DAPI, washed with PBS, and mounted using Prolong Glass Antifade Mountant.

Maegawa A., Murata K., Kuroda K., Fujieda S, & Fukazawa Y. (2022). Cellular Profiles of Prodynorphin and Preproenkephalin mRNA-Expressing Neurons in the Anterior Olfactory Tubercle of Mice. Frontiers in Neural Circuits, 16, 908964.

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Western Blot Analysis of Signaling Proteins

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Radio immunoprecipitation assay lysis buffer (Sigma‐Aldrich) was employed for protein extraction. Then, the proteins were separated by SDS‐PAGE for 2 hours and were transferred onto a PVDF membrane, which was then soaked in TBST containing 5% skim milk at 37°C for 1 hour. The primary antibodies rabbit anti‐PPP1R1B (#ab40801, 1:1000; Abcam, Shanghai, China), rabbit anti‐CREB (#ab32515, 1:1000; Abcam), rabbit anti‐CBP (#ab2832, 1 μg/mL; Abcam), and rabbit anti‐β‐Actin (#ab8227, 1:1000; Abcam) were added to the membranes, and the membranes were then incubated at 4°C overnight. After being using TBST to wash three times the membranes were subsequently incubated for 2 hours at 37°C, with goat anti‐rabbit IgG H&L secondary antibody (1:2000). The immunological reaction was detected with ECL Plus reagent (Amersham, Piscataway, NJ, USA). ImageJ 1.8.0 software was conducted to analyse data. β‐actin was employed as an internal reference protein.

Xian D, & Zhao Y. (2019). LncRNA KCNQ1OT1 enhanced the methotrexate resistance of colorectal cancer cells by regulating miR‐760/PPP1R1B via the cAMP signalling pathway. Journal of Cellular and Molecular Medicine, 23(6), 3808-3823.

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