Mice were perfused transcardially with 4% paraformaldehyde. For immunohistochemistry, the following primary antibodies were applied to 50 μm free-floating coronal OB sections: mouse monoclonal anti-tyrosine hydroxylase (TH) (1:500), anti-calretinin (CR) (1:1000) and anti-NeuN (1:200) from Millipore, mouse monoclonal anti-calbindin (CB) (1:500) from Sigma, rat monoclonal anti-BrdU (1:200), rabbit monoclonal pSer129 α-SYN (1:500) from Abcam, and rat monoclonal antibody 15G7 against human α-SYN (116–131) peptide MPVDPDNEAYEMPSEE (1:4000) from Connex31 . As secondary antibodies, we used: goat anti-mouse Alexa 488/647 (1:500), rabbit anti-rat Alexa 488/647 (1:500) from Invitrogen and biotinylated rabbit anti-rat (1:300) from Dako. Enzymatic digestion using Proteinase K (PK; Roche) was employed for 8–10 min. Peroxidase detection was applied for 8–10 min (DAB detection kit, DCS Diagnostics). Sections were imaged at 0.75 μm pixels resolution in xy dimension and 1 μm in z dimension on a confocal microscope (Zeiss LSM 510) equipped with a 10×, 25× or 40× oil objective (Zeiss). Counting of somata and nuclei was performed manually from confocal image stacks, using Zeiss AIM software. For consistent analysis, every tenth OB slice was used and imaged at predefined positions within the glomerular layer.
Mouse monoclonal anti tyrosine hydroxylase
Manufactured by Merck Group
Mouse monoclonal anti-tyrosine hydroxylase (TH) is a laboratory reagent used to detect and quantify the presence of tyrosine hydroxylase, a key enzyme involved in the biosynthesis of catecholamines such as dopamine, norepinephrine, and epinephrine. This product can be used in various immunoassay techniques, including Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA).
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Lab products found in correlation
Cy3 labelled goat anti mouse igg, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Bx51 light microscope, by Olympus (2 mentions)
Biotinylated rabbit anti rat, by Agilent Technologies (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Rat monoclonal anti brdu, by Abcam (1 mentions)
Anti calretinin, by Merck Group (1 mentions)
Anti neun, by Merck Group (1 mentions)
Proteinase k, by Roche (1 mentions)
Vectastain abc peroxidase kit, by Vector Laboratories (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Biotinylated anti mouse igg secondary antibody, by Vector Laboratories (1 mentions)
Cm1900, by Leica (1 mentions)
Dp71 ccd, by Olympus (1 mentions)
4 protocols using mouse monoclonal anti tyrosine hydroxylase
1
Quantifying Olfactory Neuron Populations
2
Tyrosine Hydroxylase Immunohistochemistry
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Immunohistochemistry was performed as described previously (Seiler et al., 2016 (link)). The brains were cut at 30 μm on a cryostat (Leica CM 1900) and mounted on Superfrost slides (Thermo Scientific). Sections were heated in citrate buffer for 30 min and blocked with 10% horse serum in 0.1% Triton-PBS. Primary and secondary antibodies were incubated in a 0.1% Triton-PBS solution containing 2.5% horse serum. Slides were incubated with the mouse monoclonal anti-tyrosine hydroxylase (TH; 1:1000, Millipore) overnight. After PBS washes, sections were incubated for 2 h with the secondary biotinylated anti-mouse IgG antibody (1:200, Vector Laboratories) and the endogenous peroxidase blocked with a solution of 10% methanol and 3% hydrogen peroxide in PBS. Thereafter, the slides were incubated with an avidin-biotin-complex (7 μl/ml; Vectastain ABC-Peroxidase KIT, Vector Labs) for 1 h and specifically bound antibodies were visualized with a metal-enhanced 3,3′-diaminobenzidine substrate kit (Pierce, 34002, Life Technologies). The sections were dehydrated in alcohol, cleared in xylene and mounted in Eukitt (O. Kindler GmbH, Freiburg, Germany).
3
Immunohistochemical Analysis of Zebrafish Alcohol Exposure
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After the adult zebrafish had been exposed to the alcohol, their eyes and brain tissues were harvested and immediately fixed in 4% paraformaldehyde, equilibrated in 30% sucrose/PBS overnight and embedded in OCT. Twelve-mm-thick sections were mounted on gelatin-coated slides and air dried at 37°C for at least 2 h. The tissue sections were rehydrated with PBS, blocked with 20% NGS and 2% BSA in 0.3% PBS/Triton X-1 00 (PBST) for 1 hr, and incubated with primary antibodies overnight at 4°C. The following primary antibodies and concentrations were used: mouse monoclonal antibody Zpr-1 (1:200, University of Oregon) for labelling cones and mouse monoclonal anti-tyrosine hydroxylase (1:400, Millipore, Billerica, MA) for labelling DA cells. The interpretation of the neuroanatomy follows the adult zebrafish brain atlas [49 ] Immunoreactions were detected using Cy3-labelled goat anti-mouse IgG diluted to 1:400 (Millipore), and the sections were counterstained with a 1:1000 dilution of 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma) to label the nuclei. The slides were examined with an Olympus BX51 light microscope (Olympus, Tokyo, Japan). The images were obtained by an Olympus CCD DP71 (Olympus) and processed using Adobe Photoshop CS (Adobe Systems, San Jose, CA).
4
Immunohistochemical Analysis of Zebrafish Eyes and Brain
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After the adult zebrafish had been exposed to the treatments for 7 days, their eyes and brain tissues were harvested and immediately fixed in 4% paraformaldehyde, equilibrated in 30% sucrose/PBS overnight and embedded in OCT. Sections of 12 μm thickness were mounted on gelatin-coated slides and air dried at 37°C for at least 2 h. The tissue sections were rehydrated with PBS, blocked with 20% NGS and 2% BSA in 0.3% PBS/Triton X-100 (PBST) for 1 h and incubated with primary antibodies overnight at 4°C. The following primary antibodies and concentrations were used: mouse monoclonal antibody Zpr-1 (1:200, University of Oregon) for labelling cones and mouse monoclonal anti-tyrosine hydroxylase (1:400, Millipore, Billerica, MA) for labelling DA cells. The interpretation of neuroanatomy follows the adult zebrafish brain atlas36 . Immunoreactions were detected using Cy3-labelled goat anti-mouse IgG diluted to 1:400 (Millipore). The sections were counterstained with a 1:1000 dilution of 4',6-diamidino-2-phenylindole (DAPI) (Sigma) to label the nuclei. The slides were viewed with an Olympus BX51 light microscope (Olympus, Tokyo, Japan). The images obtained by an Olympus CCD DP71 (Olympus) were processed using Adobe Photoshop CS (Adobe Systems, San Jose, CA).
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