For Western blot, mice were perfused transcardially with PBS to remove blood. The brains were collected and different brain regions were dissected on ice. All the samples were stored at −80 °C. The brainstem samples were homogenized in cold RIPA lysis buffer containing proteinase and phosphatase inhibitors.28 (link),36 (link) Equivalent amounts of protein from each group were added to a 4–12% Bis-Tris-polyacrylamide electrophoresis gel. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. We blocked the membranes with 5% skim milk and then incubated them with primary antibodies against TH (1:1000, ab129991, Abcam, Cambridge, MA, USA), Iba-1 (1:1000, 019–19,741, Abcam, Cambridge, MA, USA), CR3 (1:1000, ab128797, Abcam, Cambridge, MA, USA) or GAPDH (1:5000, ab181602, Abcam, Cambridge, MA, USA) at 4 °C overnight. The membranes were then washed three times with PBST and incubated with horseradish peroxidase-linked anti-rabbit IgG (1:3000) antibody at room temperature for 2 h. The blot signals were detected by ECL reagents (Biological Industries, Cromwell, CT, USA). The densitometry of blots was quantified based on previous report.46 (link) GAPDH was used as an internal control for each blotting. All blots were normalized to GAPDH. Fold changes for each treatment were normalized and are shown as percentages of the control.
Ab129991
Manufactured by Abcam
Sourced in United States
Ab129991 is a laboratory instrument used for the analysis and quantification of various biomolecules. It is a highly sensitive and precise tool that employs advanced detection techniques to provide accurate measurements. The core function of this product is to facilitate the study and investigation of biological samples, supporting researchers in their scientific endeavors.
Automatically generated - may contain errors
Lab products found in correlation
Ab128797, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ecl reagent, by Sartorius (1 mentions)
Ab97041, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Ab18207, by Abcam (1 mentions)
Ab14849, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Ab92434, by Abcam (1 mentions)
Fluoromount g mounting medium, by Southern Biotech (1 mentions)
Imaris 9, by Oxford Instruments (1 mentions)
Nis elements 5, by Nikon (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Ab18723, by Abcam (1 mentions)
Ab105389, by Abcam (1 mentions)
Ab79351, by Abcam (1 mentions)
Inverted fluorescence microscope, by Leica (1 mentions)
6 protocols using ab129991
1
Western Blotting Analysis of Brain Samples
2
Flow Cytometry of Neural Cell Markers
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FACS was performed as previously reported [1 (link),2 (link)]: The primary antibodies used were anti-tyrosine-hydroxylase (TH) monoclonal antibody (1:100, ab129991, Abcam) and anti-βIII-tubulin mouse monoclonal antibody (1:500) The secondary antibodies used were goat anti-mouse IgG H&L phycoerythrin (1:500, ab97041, Abcam) and Alexa Fluor® 488-conjugated goat anti-mouse (1:500, A11001, Molecular Probes). The cells were analyzed by FACS Verse (BD Bioscience, San Jose, CA, USA), using FACS suiteTM software (BD Bioscience). FACS analyses were repeated in triplicate.
3
Immunocytochemical Characterization of Dopaminergic and Astrocytic Cultures
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DA neurons or astrocytes in cultures were seeded on 20 mm diameter glass coverslips that were individually placed in each well of 12 well plates and were fixed by 4% paraformaldehyde (PFA) for 10 min at room temperature. Cells were then washed and first permeabilized with 0.1% Triton X-100 and blocked in 10% BSA in PBS for 30 min before staining. The cells were incubated overnight at 4 C° with the following primary antibodies: mouse anti-tyrosine hydroxylase (TH) (1:1000, Abcam, ab129991), rabbit anti-class III beta-tubulin (Tuj1) (1:1000, Abcam, ab18207), rabbit anti- glial fibrillary acidic protein (GFAP) (1:2000, Abcam, ab7260), and mouse anti-S100b (11,000, Abcam, ab14849). Then the detection of target antigen was performed by species-appropriate fluorescence-conjugated secondary antibodies Alexa flour 488, 555, or 647. Cells were visualized and imaged by a Zeiss Confocal Microscope.
4
Immunofluorescent Staining of Dopaminergic Neurons
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Coverslips with DA neurons were fixed in 4% paraformaldehyde (PFA) for 15 min at 37 °C. After washing with DPBS, the cells were blocked and permeabilized in a solution of DPBS, 0.2% Molecular Grade Triton X-100, and 10% Donor Horse Serum for one hour. Primary antibodies were added to the blocking solution at 4 °C overnight, with the following dilutions for DA neurons: TH (Abcam ab129991) (1:500) and MAP2 (Abcam ab92434) (1:500) (please note that while MAP2 is mainly expressed in neurons, there may be some low expression in glial cells). The next day, the coverslips were washed with DPBS and incubated with Alexa Fluor secondary antibodies, followed by counterstaining with DAPI staining solution (1:3000) for one hour at room temperature. The coverslips were rewashed, mounted on slides using Fluoromount-G mounting medium (0100-01, Southern Biotech), and allowed to dry overnight in the dark. The fluorescence signals were visualized using a Nikon A1-R confocal microscope, and images were processed using NIS elements 5.21 (Nikon) and microscopy image analysis software Imaris 9.8 (Oxford Instruments).
5
Immunofluorescence of Dopaminergic Neurons
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DA neurons were seeded on 20-mm diameter glass coverslips that were individually placed in each well of 12-well plates and fixed in 4% PFA for 10 min at room temperature. Cells were washed and permeabilized with 0.1% Triton X-100 and blocked in 10% BSA in PBS for 30 min before staining. The cells were incubated overnight at 4°C with the following primary antibodies: mouse anti-TH (1:1,000, ab129991; Abcam), rabbit anti-nestin (1:1,000, ab105389; Abcam), mouse anti-SOX2 (1:1,000, ab79351; Abcam), and mouse anti-DCX (1:2,000, ab18723; Abcam). The detection of target antigen was performed by species-appropriate Alexa Fluor 488 or 555-conjugated secondary antibodies. Cells were visualized and imaged by a Leica inverted fluorescence microscope.
6
Protein Expression Analysis in Rat Tissues
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Each sample of NG or NTS, LVAW, and TA tissue was extracted from 3–4 rats or 1 rat, respectively. The total protein was incubated at 4°C for 1 h in RIPA buffer containing 1% protease inhibitor. The protein extracts were separated on 12% SDS‐PAGE and transferred to nitrocellulose membranes, which were further blocked with 5% non‐fat dry milk for 2 h, and then incubated at 4°C overnight with the primary antibodies anti‐GAPDH (Abcam Cat # G8795), anti‐β‐actin (Wanleibio, WL01845), anti‐tyrosine hydroxylase (Abcam, ab129991), and anti‐α‐synuclein (Abcam, ab27766), respectively, followed by incubation with the appropriate secondary antibodies (anti‐rabbit/anti‐mouse) at room temperature for 50–60 min. Specific antibody‐antigen complexes were detected using the Odyssey Infrared Imaging System (LI‐COR Biosciences).
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