The phenotypes of proerythroblast (ProE), erythroblast A (EryA), erythroblast B (EryB), and erythroblast C (EryC) were determined by flow cytometry by immunostaining with specific antibodies. The strategy of gating erythroblasts at different stages was used as described previously (Koulnis et al., 2011 ). Four stages of erythroblasts were defined as: immature, large “ProE” erythroblasts: CD71highTer119intermediate; less mature, large “EryA” erythroblasts: CD71highTer119highFSChigh; smaller, more mature “EryB” erythroblasts: CD71highTer119highFSClow; mature “EryC” erythroblast subset: CD71lowTer119highFSClow. The specific monoclonal antibodies against the antigens or isotype-matched IgG controls used were: Fc blocker: CD16/CD32 (FCR4G8, eBioscience, Thermo Fisher Scientific, US), CD71 (C2F2, BD Pharmingen, US), Ter119 (Ter119, BD Pharmingen), DAPI (Sigma-Aldrich, US). For intracellular staining, cells were treated with intracellular fixation and permeabilization Buffer (eBioscience, Thermo Fisher Scientific), and anti-ChAT antibody (ab181023, abcam). The stained cells were detected using a Fortessa flow cytometer (BD Pharmingen) and analyzed with FlowJo 10.1 software (BD Biosciences, US). The frequency (%) of total cells was presented in all the experiments.
Ab181023
Manufactured by Abcam
Sourced in United Kingdom
Ab181023 is a primary antibody. It is a recombinant monoclonal antibody raised against a specific target. The core function of this product is to detect and bind to its target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Ter119, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Cd16 cd32 fcr4g8, by Thermo Fisher Scientific (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Bca protein assay kit, by Wuhan Servicebio Technology (1 mentions)
Sa5 35571, by Thermo Fisher Scientific (1 mentions)
Ab216485, by Abcam (1 mentions)
Gb15003, by Wuhan Servicebio Technology (1 mentions)
Sab4200559, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Dapi c1005, by Beyotime (1 mentions)
Alexa fluor 594 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 sr, by Leica (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, by Beyotime (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Ab181086, by Abcam (1 mentions)
Ab177487, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
8 protocols using ab181023
1
Erythroblast Phenotyping by Flow Cytometry
2
Cerebral Cortex and Gastric ChAT Expression
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A Western blot analysis was performed to assess the expression of ChAT protein in the cerebral cortex on the infarcted side and in gastric tissues. The total protein concentration in each group was determined using the BCA Protein Assay Kit (G2026-200T, Servicebio). Subsequently, 10 μg of each sample was loaded onto a 10 % SDS-PAGE gel for electrophoresis. Following electrophoresis, the proteins were transferred onto PVDF membranes (IPVH00010, Millipore) and blocked with 5 % skim milk for 1 h at room temperature. The membranes were then incubated overnight at 4 °C on a shaker with primary antibodies, including ChAT (1:1000, ab181023, Abcam), α7nAchR (1:1000, ab216485, Abcam), and β-actin (1:1000, GB15003, Servicebio). On the following day, the membranes were incubated with a fluorescent secondary antibody (1:10000, SA5-35571, Invitrogen) and visualized using an Odyssey imager.
3
Western Blot Analysis of Brain Proteins
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Brain homogenates were extracted with RIPA lysate for Western blot analysis. The protein concentrations were quantified using the BCA method to 5 μg/μL, and the protein lysates were separated using SDS‐PAGE methods. The proteins were transferred to the PVDF membrane (Merck Millipore) through a wet‐transfer protocol, and the membranes were blocked with 5% skimmed milk for 1 h at room temperature and washed in TBST for 3 × 5 min. The membranes were incubated with primary antibodies at 4°C: Rab5a (1:1000; 24 h, CST, E6N8S); Rabep1 (1:5000; 24 h, Abcam, ab176578); TrkA (1:300; 36 h, Abcam, ab216626); pTrkA (1:500; 36 h, Invitrogen, PA5‐37672); AKT (1:1000; 24 h, CST, #4691); pAKT (1:1000; 36 h, CST, #4060); ERK (1:5000; 24 h, Abcam, ab184699); pERK (1:2000; 24 h, CST, #4370); ChAT (1:5000; 24 h, Abcam, ab181023); AchE (1:5000; 36 h, Abcam, ab183591); vAchT (1:1000; 36 h, Sigma, sab4200559); ChT1 (1:5000; 24 h, Abcam, ab154186); m1AchR (1:1000; 36 h, boster, BA1543); m2AchR (1:5000; 24 h, Abcam, ab109226); GAPDH (1:5000; 24 h, Proteintech, 60,004–1‐1 g); and β‐actin (1:5000; 24 h, Proteintech, 66,009–1‐1 g). Following primary antibody incubation, the membranes were washed in TBST for 3 × 5 min and incubated with HRP‐conjugated secondary antibody (1:5000) for 1 h at room temperature. The protein bands were visualized and imaged by the Bio‐Rad ChemiDoc Imaging System.
4
Medulla Oblongata ChAT Expression Analysis
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After sacrifice, the rats were rapidly perfused with 4 % paraformaldehyde, and the medulla oblongata was carefully extracted. The extracted tissues were embedded in optimal cutting temperature and 10-μm sections were obtained, after which they were sealed with 5 % bovine serum albumin at room temperature for 1 h. Subsequently, the sections were incubated with a ChAT primary antibody (1:500, ab181023, Abcam) overnight at 4 °C. The following day, Alexa Fluor 594 secondary antibody (1:500, A-11012, Invitrogen) was added and incubated at room temperature in the dark. DAPI (C1005, Beyotime) was used to label the cell nuclei. Finally, the DMV on the infarcted side was examined at 100 × magnification using a fluorescence microscope (TCS SP8 SR, Leica).
5
Quantifying Cholinergic Markers in Rat PFC
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Animals were sacrificed after MWM, and the brains were harvested. PFC tissue was homogenized with a tissue grinder in RIPA (Beyotime Biotechnology, China), containing a phosphatase and protease inhibitor cocktail, followed by centrifugation of the homogenized tissue samples at 12,000 g for 15 min at 4°C. The supernatants were collected, and their protein concentrations were measured using the Thermo protein assays (Thermo, USA). Samples (20 μg) were electrophoresed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels (Beyotime Biotechnology, China), and the separated proteins were transferred at 300 mA for 70 min onto nitrocellulose membranes (pore size, 0.45 μm, Millipore, USA). The membranes were blocked for 1 hr with Tris‐buffered saline with Tween‐20, containing 3% bovine serum albumin (BSA), followed by incubation with primary antibodies [choline acetyltransferase: ChAT (1:1,000 dilution, number ab181023">ab181023 , abcam, USA); vesicular acetylcholine transporter: VAChT (1:1,000 dilution, number ab134298, abcam, USA); acetylcholinesterase: AChE (1:1,000 dilution, NB1‐59170, NOVUS, USA); high affinity choline transporter: ChT (1:1,000 dilution, number PA5‐42485, Thermo, USA)] and the appropriate secondary antibodies coupled to horseradish peroxidase.
6
Immunofluorescence Quantification of Spinal Cord Markers
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Immunofluorescence was performed for the measurement of PV-positive interneurons, matrix metalloproteinase-9 (MMP-9), choline acetyltransferase (ChAT), neuronal marker neuronal nuclei (NeuN) and ErbB4. Briefly, the spinal cord was collected, fixed with 4% paraformaldehyde, embedded with paraffin, sliced, dewaxed and hydrated with ethanol. The citric acid buffer was then added, following with PBS wash for three times. After blocked with 5% bovine serum albumin (BSA) for 30 minutes at 37°C, the samples were incubated with the primary antibodies of anti-PV (1/250, ab181086, Abcam, UK), anti-MMP-9 (1/5000, ab228402, Abcam), anti-ChAT (1/100, ab181023, Abcam), anti-NeuN (1/100, ab177487, Abcam) and anti-ErbB4 (1/500, ab19391, Abcam) overnight at 4°C. Then, samples were incubated with the corresponding secondary antibody (ab205719, Abcam) for 45 minutes at 37°C. DAPI was used for redyeing nucleus. A CKX53 laser scanning confocal microscope (Olympus, Japan) was used to take the photomicrographs. Briefly, every sixth section covering all the target cells was included (7~8 sections per animal), counted by a 400× lens. The cell ratio was determined using ImageJ software (Rasband, NIH, USA) through the calculation of cell areas. The atlas of spinal cord of SOD1G93A C57BL/6J mice was used as a reference.
7
Western Blot Analysis of ChAT Expression
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Tissues from the NAc were homogenized in RIPA buffer and Western blotting was performed as previously described [19] . Primary antibodies were against choline acetyltransferase (ChAT, 1:3000, ab181023, Abcam, Cambridge, UK) and β-actin (1:1000, bsm-33036M, Bioss, Beijing, China). The secondary antibodies were anti-rabbit and anti-mouse (1:5000, 926-32211and 926-32212, Li-cor, Lincoln, NE, USA) antibodies. Immunoreactive protein bands were detected by using the Odyssey CLx infrared imaging system. The darkness of the blots was evaluated by analysis of the intensity of each band using ImageJ software (Version 1.8.0, NIH Image, Bethesda, MD, USA). Data were expressed as ratios of optical density (OD) compared with controls (β-actin) for statistical analysis.
8
Immunohistochemical Characterization of Cholinergic and Dopaminergic Receptors
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Coronal slices were prepared and blocked for 1 h at room temperature in 1% albumin and 0.5% Triton-X in PBS. Slices were then incubated overnight at 4 °C in 1% albumin in PBS with the following antibodies: anti-ChAT (1:200, ab181023, Abcam, Cambridge, UK); antidopamine receptor 1 (D1R, 1:100, Ab20066, Abcam, Cambridge, UK) and anti-dopamine receptor 2 (D2R, 1:100, sc-5303, Santa Cruz, Dallas, TX, USA). Sections were then dark incubated 1 h at room temperature in blocking buffer with AlexaFluor 488 donkey anti-goat IgG (1:400, ab150077, Abcam, Cambridge, UK), AlexaFluor 555 donkey anti-mouse IgG (1:400, ab150106, Abcam, Cambridge, UK) and AlexaFluor 647 donkey anti-rabbit IgG (1:400, ab150075, Abcam, Cambridge, UK). Sections were washed with PBS (×3, 5 min each) after each incubation step. Samples were visualized using the Leica SP8 laser scanning confocal microscope and Leica LAS X image acquisi- tion software. Quantitative analysis (cell counts) was performed using LAS X software (Version 2.0.1, Mannheim, Germany).
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