To extract the total protein from each sample, the PRO-PREPTM kit (American Intron Biotechnology) was used. After, the total amount of each protein was quantified under the guidelines of Bradford Protein Analysis (Bio-Rad, CA, USA) and protein samples were kept at −80°C until they were used in the analysis. 30 μg of total protein extracted from each sample was separated on a 13% sodium dodecyl sulphate-polyacrylamide gel and transferred to a Immunoblot polyvinylidene fluoride membranes (Bio-Rad, USA). It was then detected using secondary antibody (anti-rabbit and/or anti-mouse secondary antibody; diluted 1:5,000; Abcam, MA) after inducing antigen antibody reactions using MC1R (diluted 1:1,000; TA308794, OriGene, USA, MD), TYR (diluted 1:1,000; AB6211, Abcam, UK, Cambridge) and β-actin (diluted 1:5,000; AB49900, Abcam) in the membrane where the protein was transferred. The membrane was then fluorescently reacted with ECL and analyzed after 1–5 min of exposure in diagnostic films.
Ab6211
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab6211 is an antibody product manufactured by Abcam. It is a recombinant monoclonal antibody that recognizes an epitope on the human FRK protein. The antibody is intended for use in applications such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab214672, by Abcam (3 mentions)
Bradford protein analysis, by Bio-Rad (1 mentions)
Immunoblot polyvinylidene fluoride membranes, by Bio-Rad (1 mentions)
Ab49900, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mab360, by Santa Cruz Biotechnology (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Ab190195, by Abcam (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Immobilon western ecl system, by Bio-Rad (1 mentions)
Ab8227, by Abcam (1 mentions)
Lsm 700, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Triton x, by Merck Group (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Ab36001, by Abcam (1 mentions)
Tnb blocking solution, by PerkinElmer (1 mentions)
Ab16053, by Abcam (1 mentions)
Ab6199, by Abcam (1 mentions)
19 protocols using ab6211
1
Protein Extraction and Quantification
2
Immunofluorescence Staining of Brain and Cell Samples
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For immunofluorescence of brain slices, the sections were blocked with 5% FBS in PBST (0.3% Triton X-100 in PBS) for 1 h, followed by overnight incubation with primary antibody at 4°C. The sections were then washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit (1:1,000, Invitrogen, A11008) or Alexa Fluor 555 goat anti-mouse (1:1,000, Invitrogen, A21422) for 1 h at room temperature. The sections were ultimately rinsed with PBS and then mounted onto adhesive slides. Fluorescently labeled sections were visualized using an Olympus scanning microscope (Olympus BX51, Japan). For immunocytochemical staining, primary cells were rinsed with 0.1 M PBS and fixed with 4% paraformaldehyde for 20 min. Cell cultures on the cell slides were then prepared using the same procedures for the immunofluorescence of the brain slices.
The primary antibodies used for immunofluorescent staining were as follows: rabbit anti-TH antibody (1:1,000, abcam, ab6211), rabbit anti-GFAP antibody (1:1,000, abcam, ab7206), rabbit anti-Iba-1 antibody (1:1,000, Wako, 019-19741), rabbit anti-NeuN (1:100, CST, 24307), mouse anti-kir6.2 antibody (1:200, Santa Cruz, sc-390104), rabbit anti-C3 antibody (1:100, abcam, ab11887), mouse anti-GFAP antibody (1:1,000, Millipore, MAB360), mouse anti-Drp1 (1:200, Santa Cruz, sc-101270) and rabbit anti-TOM20 (1:200, Proteintech, 11802-1-AP).
The primary antibodies used for immunofluorescent staining were as follows: rabbit anti-TH antibody (1:1,000, abcam, ab6211), rabbit anti-GFAP antibody (1:1,000, abcam, ab7206), rabbit anti-Iba-1 antibody (1:1,000, Wako, 019-19741), rabbit anti-NeuN (1:100, CST, 24307), mouse anti-kir6.2 antibody (1:200, Santa Cruz, sc-390104), rabbit anti-C3 antibody (1:100, abcam, ab11887), mouse anti-GFAP antibody (1:1,000, Millipore, MAB360), mouse anti-Drp1 (1:200, Santa Cruz, sc-101270) and rabbit anti-TOM20 (1:200, Proteintech, 11802-1-AP).
3
Dual Immunohistochemistry of PVN and RVLM
4
Western Blot Analysis of Brain Proteins
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The brain tissue was homogenized using a tissue grinder (Kimble) in 1.5 ml tubes containing lysis buffer composed of 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA pH 8.0, 1% SDS, and a protease inhibitor cocktail. Subsequently, the homogenates were centrifuged at 14,000 × g for 10 minutes at 4 °C. The supernatant was collected and heated in a dry bath at 75 °C for 20 minutes to denature proteins. The denatured tissue lysates were then resolved using SDS-PAGE (Bio-Rad) and transferred onto nitrocellulose membranes. Following a 1-hour block in Tris-buffered saline with 5% non-fat milk and 0.5% Tween-20 (TBST), membranes were incubated overnight at 4 °C with primary antibodies. The next day, after three washes with TBST, membranes were incubated at room temperature for 1 hour with HRP-conjugated secondary antibodies (CWS) in TBST containing 5% milk powder. Subsequently, membranes were washed three times with TBST. Bands were visualized using the Immobilon Western ECL system (Bio-Rad, USA), and data analysis was performed using Gel-Pro Analysis (Media Cybernetics, USA). Anti-β-actin antibody (Abcam, ab8227, 1:1000), anti-c-Fos antibody (Abcam, ab214672, 1:500), anti-Tyrosine Hydroxylase antibody (Abcam, ab6211, 1:500) were used. All the uncropped blots were supplied in the Source data file.
5
Visualizing Catecholaminergic Neurons
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Mice were anaesthetized with ketamine/xylazine solution and perfused transcardially with heparinized saline followed by fresh fixative solution (paraformaldehyde (PFA, 4%) in 0.1 M phosphate buffer). The brains were collected, and cut into coronal 25 μm sections using a cryostat, sections were then collected into tubes containing tissue storage solution consisting of 50 ml glycerin, 50ml ethylene glycol and 100 ml 0.1 M phosphate buffer (pH 7.5) and stored until use in 4°C. The sections were washed (3 x 15 min) in TNT with Triton-X (0,1%) (Sigma-Aldrich St.Louis, MO, USA). For tyrosine hydroxylase (TH) visualization , the sections were incubated for two days in TNB blocking solution (Perkin Elmer, Akron, Ohio, USA) with 1:2000 Goat polyclonal antibody to TH (ab6211, Abcam, Cambridge, UK). The sections were then washed in TNT with Triton-X (0,1%) and incubated in TNB blocking solution with 1:1000 Donkey anti-goat Alexa Fluor 568 (ab36001, Abcam). The cell nuclei were stained with DAPI (1:5000; Life Technologies, Carlsbad, CA, USA). The sections were then washed in TNT (2 x 15 min), submerged in 0.1M PB and mounted on microscope slides (Superfrost Plus, Menzel) together with ProLong Gold Antifade (Life Technologies). The GLP-1 fibers were visualized with a confocal microscope (LSM 700; Carl Zeiss, Oberkochen, Germany).
6
Immunohistochemical Quantification of Neuronal Proteins
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Sections were incubated with 3% H2O2 for 15 min, then blocked with goat serum protein (Solarbio, Beijing, China) for 30 min at 37°C. After pouring slowly, it was incubated with the corresponding primary antibody overnight at 4°C. The primary antibody was presented as follows: rabbit polyclonal to NGF (1 : 1000; ab6199, Abcam, Cambridge, MA, USA), TH (1 : 800; ab6211, Abcam), CHAT (1 : 1000; ab6168, Abcam), and GAP43 (1 : 1000; ab16053, Abcam). After incubation at 37°C for 1 h and washing with 0.1 M PBS three times, secondary antibodies (1 : 3000; Abcam) were added to incubate for 20 min. After washing sections, it was visualized using the DAB reagent. Nuclear counter stain hematoxylin was dehydrated with ethanol and mounted with glycerol gelatin. Images were measured using ImageJ 6.0 system.
7
Immunofluorescent Visualization of VAChT and TH
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Frozen sections were blocked with 1% BSA for 2 h, incubated with anti-vesicular acetylcholine transporter (VAChT) antibody (ab68984, Abcam, Cambridge, UK, dilution 1:300), anti-tyrosine hydroxylase (TH) antibody (ab6211, Abcam, Cambridge, UK, dilution 1:500) at 4 °C overnight, and then incubated with AlexaFluor488-conjugated secondary antibody at 37 °C for 1 h. Nuclei were stained with DAPI. Confocal images of 10 μm Z-projection were captured by a laser scanning confocal microscope (Leica TCS SP8, Wetzlar, Germany).
8
Stereological Quantification of Midbrain Cells
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The number of TH- and GFAP-positive cells in the substantia nigra was estimated using a random sampling stereological counting method. Images were sampled from at least four different points within each substantia nigra section [37 (link)]. TH (ab6211), GFAP (ab7260), and DAPI (ab104139) were from Abcam, USA. All immunoreactive cells were counted regardless of the intensity of labeling. The slides were photographed with a fluorescence microscope (Nikon, Japan).
9
Immunofluorescence Labeling of Chromaffin Cells
10
Quantifying Tyrosine Hydroxylase Neuron
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Tyrosine hydroxylase (TH)-positive cells were detected by IHC. Brain tissue slices (5 µm) were incubated with 3% H2O2 to remove endogenous peroxidase activity, followed by blocking with blocking buffer. Slices were incubated with anti-TH antibodies (ab6211, 1; 1000, Abcam) overnight at 4 °C and then incubated with secondary antibodies rabbit IgG (ab205718). Next, slices were incubated DAB (D3939, Sigma-Aldrich, USA). The image was captured using a light microscope (Leica DM4000) at × 100 magnification. Finally, the results were analyzed by counting the number of positive cells on a bright microscope.
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