Triformol

Brinzolamide (BRZ) (China Chen Xi Pharmaceutical Institute, batch number: 201503); PEG (medical grade, Hebei Huanghua Xinnuolixing chemical stock corporation, average relative molecular weight: 2000 Da); SA (Sigma-Aldrich, St. Louis, MO); 1% tropicamide eye drops (Santen, Japan); anesthetic drop TobraDex (Alcon, USA); rabbit polyclonal Tuj-1 antibody (1:100, Proteintech, Rosemont, IL); 5% chloral hydrate, 4% triformol, penbritin, and DAPI (40,6-diamidino-2-phenylindole) (Sigma-Aldrich, St. Louis, MO); hematoxylin solution (M type) and 0.3% eosin alcoholic solution (Muto Pure Chemicals, Tokyo, Japan); mounting medium (Vectashield, Vector Laboratories Inc., Burlingame, CA). The miRNA expression plasmids for miRNA 124, pcDNA 3.2/V5-hsa-rni-124a were purchased from Addgene (Addgene plasmid 26306), and other chemical reagents were analytically pure.

Cells were transfected with the indicated constructs for 72 h. Then, the cell suspension containing 1 × 10⁴ cells was seeded in the uncoated (for migration assay) or coated (for invasion assay) top chambers with Matrigel (BD Biosciences, USA). The RPMI‑1640 medium containing 20% FBS were added to the low chamber. After being incubated for 24 h, these cells were fixed with 4% triformol (Sigma Aldrich, USA) and stained with 1.5% crystal violet (Solarbio, Beijing, China) at 37°C. Migrated and invaded cells were determined and quantified with an inverted microscope (Leica, Germany) [44 (link)].

According to a stereotaxic atlas of rats, 3 mm of the anterior brain, containing the frontal lobe, and 5 mm of the middle section, containing the hippocampus and hypothalamus, were removed. The removed cerebral tissues were fixed in triformol (Sigma-Aldrich, St. Louis, MO, USA) for 1 week at a temperature of 4°C. Samples were dehydrated in a gradient series of hydrous ethanol and were then treated with xylene to make the samples transparent. Dehydrated samples of cerebral tissues were embedded in paraffin and then sliced by microtome (RM2015; Leica, Wetzlar, Germany) at a thickness of 5 μm. After deparaffinizing and hydration treatments, the sections were again washed with PBS three times, before being incubated with primary antibodies overnight at a temperature of 4°C. The primary antibodies included goat polyclonal antibodies to the 5-HT_3A receptor (Abcam, Cambridge, MA, USA; ab51950, 1 : 500) and to the 5-HT_3B receptor (Abcam; ab115023, 1 : 50). After washing, the sections were incubated with secondary antibodies (Cy3-labeled donkey anti-goat IgG (H+L); Beyotime, Haimen, China; A0505, 1 : 2000; and Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L); Beyotime, A0423, 1 : 2000) for 2 h. A laser scanning confocal microscope (ZEISS LSM710) was then used to observe the samples [26 (link), 27 (link)].