pitx3 sc 19307  (Santa Cruz Biotechnology)

Journal: Neurochemical Research

Article Title: MicroRNA-124-3p Modulates Alpha-Synuclein Expression Levels in a Paraquat-Induced in vivo Model for Parkinson’s Disease

doi: 10.1007/s11064-024-04130-y

Figure Lengend Snippet: Effects of miR-124-3p in the expression of dopaminergic neuronal markers in the SN and striatum of a rat PQ model in vivo. (a) Design and timeline of the experimental animal procedure. Male Wistar rats were subjected to intranigral injection with 250 nM miR-124-3p, or saline, followed by four i.p injections of saline or PQ (10 mg/kg), separated by one day. Five days after the last PQ injection, the brains were collected for western blot analysis. Expression levels of TH in the (b) SN and (c) striatum, and (d) PITX3 in the SN of adult Wistar rats treated with saline, PQ or 250 nM miR-124-3p + PQ. (e) Representative western blot protein bands of PITX3 (32 kDa) and actin (42 kDa). TH and PITX3 protein levels were normalized against actin. Data are expressed as a percentage of the controls (saline) ± SEM. Protein expression in the control condition (saline) was set to 100%. N = 3 or 4 rats. * p < 0.05 compared to the saline group and ## p < 0.01 compared to the PQ-treated group using the one-way ANOVA followed by Sidak’s multiple comparison test. Abbreviations: i.p, intraperitoneal; TH, tyrosine hydroxylase; PITX3, pituitary homeobox 3; PQ, paraquat; SN, substantia nigra

Article Snippet: Then, 30 µg of total protein was loaded in a 12.5% or 15% polyacrylamide gel, followed by electrophoresis and transfer onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Little Chalfont, UK) for 90 min. Membranes were blocked using 5% milk or 5% bovine serum albumin and then incubated with the following primary antibodies: mouse anti-TH (1:5000; BD Biosciences), rabbit anti-PITX3 (1:1000; Invitrogen), mouse anti-α-synuclein (1:500; BD Biosciences); rabbit anti-pS129-α-synuclein (1:200; Santa Cruz Biotechnology), goat anti-NOX1 (1:200; Santa Cruz Biotechnology), mouse anti-Rac1 (1:750; Millipore), rabbit anti-p47 phox (1:1000; Santa Cruz Biotechnology), mouse anti-actin (1:5000; BD Biosciences) and mouse anti-beta-tubulin (1:5000; BD Biosciences); and the secondary antibodies conjugated to horseradish peroxidase—HRP (chicken anti-goat, goat anti-rabbit or goat anti-mouse; 1:5000; all from Santa Cruz Biotechnology).

Techniques: Expressing, In Vivo, Injection, Saline, Western Blot, Comparison

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA injection decreases dopaminergic neurons without affecting β-III Tubulin. (A) Coronal sections of the ventral midbrain from E14 vehicle- and HA-injected rat embryos stained to detect neurons (β-III Tubulin+) and dopaminergic phenotype (TH+); a representative section of a vehicle-injected embryo showing many TH-positive neurons while in a similar midbrain section from a representative HA-injected embryo less TH-positive cells are present. Nuclei were detected with Hoechst. Inner white squares in the merge images represent the higher magnifications shown at the bottom. Scale bars: 150 μm and 50 μm for the low-power and high-power pictures, respectively. (B) β-III Tubulin and TH expression by qRT-PCR from E14 vehicle- and HA-injected embryos, with β-III Tubulin presenting no change, and a significant decrease in TH caused by HA. **p < 0.01. (C) Densitometric analyses of the effect of HA administration on protein levels of β-III Tubulin and diverse factors involved in dopaminergic specification and phenotype. HA injection decreased the protein level of TH, Lmx1a, Lmx1b and Pitx3 in midbrain tissue from E14 HA-injected embryos without affecting the generation of neurons, compared to the vehicle-injected condition. Values were normalized to GAPDH signal. *p < 0.05.

Article Snippet: The following antibodies were used: rabbit anti-rat H 1 R polyclonal antibody (1:1500, Santa Cruz Biotechnology); goat anti-rat H 2 R polyclonal antibody (1:1500, Santa Cruz Biotechnology); rabbit anti-Lmx1a polyclonal antibody (1:100, Abcam); rabbit anti-Lmx1b polyclonal antibody (1:100, Abcam); rabbit polyclonal anti-tyrosine hydroxylase (TH, 1:1000; Pel-freez) and rabbit anti-Pitx3 polyclonal antibody (1:500, Zymed).

Techniques: Injection, Staining, Expressing, Quantitative RT-PCR