Cells were collected at different times. Cells were seeded in 10 cm dishes and then transfected and induced as described above. The cells were then lysed in a lysis buffer containing RIPA and an inhibitor cocktail (1:10). Cell lysates containing 30 μg of protein were resolved by SDS-PAGE and then transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membrane was then blocked with 5% skim milk, followed by incubation with the following primary antibodies overnight at 4°C: rabbit anti-human Cav-1 (1:2000; Abcam), rabbit anti-human Lmx1a (2 μg/mL; Abcam), rabbit anti-human Nurr1 (1:100; Santa Cruz Biotechnology), rabbit anti-human TH (1:1000; Abcam) or rabbit anti-human β-actin (1:2000; Abcam). After four washes with Tris-buffered saline/Tween, the membrane was incubated with secondary antibody for 1.5 hours at room temperature. Protein signals were detected with the ECL kit (Beyotime, Shanghai, China) and quantitatively analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Rabbit anti human th
Manufactured by Abcam
Sourced in Japan
Rabbit anti-human TH is a primary antibody that recognizes the tyrosine hydroxylase (TH) protein, a key enzyme involved in the biosynthesis of catecholamine neurotransmitters such as dopamine, norepinephrine, and epinephrine. This antibody is raised in rabbits and is specific for the human TH protein.
Automatically generated - may contain errors
2 protocols using rabbit anti human th
1
Western Blot Analysis of Protein Expression
2
Immunohistochemical Analysis of UCP1, TH, and vAChT
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Sections were used for immunohistochemical analysis of UCP1, TH, and vAChT. Immunoreactivity was assessed according to the enzyme antibody technique with the following steps: endogenous peroxidase blocking with 3% hydrogen peroxide in methanol for 20 min at room temperature (RT), 5% normal goat serum in PBS (pH 7.2), 30 min at RT to block non-specific sites, and rabbit anti-human UCP1 (1/1000, Abcam Inc.), rabbit anti-human TH (1/10000, Abcam Inc.), or rabbit anti-human vAChT (1/10000, MBL, Nagoya, Japan) overnight at 4°C. The EnVision system with labeled polymer–HRP (30 min at RT) and DAB and 30% hydrogen peroxide in 0.05 M Tris-HCL buffer (pH 7.6, 10 min at RT) were used to identify positive signals.The sections were finally counterstained with hematoxylin.
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