Hoechst 33258

An oxidative fluorescent dye hydroethidine (Polyscience Inc., Warrington PA, USA) was used for semi-quantitative evaluation of superoxide in situ [15 (link)]. Hydroethidine (7 mg) was diluted with N,N-dimethylacetamide (1 mL). The whole brain was quickly frozen at −80°C. Twenty-μm-thick coronal sections of the brain were cut on a cryostat and mounted onto microscope slides [18 ]. Each slice was incubated with hydroethidine (2×10⁻⁶ mol/L) in a light-protected chamber at 37°C for 20 min [15 (link)]. Hoechst 33258 (1 μg/mL, Nacalai Tesque, Kyoto, Japan) was simultaneously applied to stain nuclei of cells. Images of cellular fluorescence were acquired using a microscope fitted with BZ-II analyzer software (Model BZ-9000 Generation II, Keyence, Osaka, Japan). Settings were adjusted based on the fluorescence intensity in tissues from the Control group and were identical for the acquisition of images from all of the tissues. The negative control did not show any nonspecific staining. The total ethidium bromide fluorescence was determined by subtracting that of background in each specimen. Six fields of view were analyzed using three sections from different animals (two fields from each) of the hippocampus CA1 region.

Frozen tissues in OCT chemical compound were sliced into sections (30 μm) on one CM3000 cryostat, permeabilized into 0.3% Triton X-100/PBS solution and hatched using primary antibodies against miR-30d-5p, Ki-67 (1:200, Osaka, Japan), SERPINE1 (1:200, Santa Cruz, USA), LCAD (1:500, Shanghai, China), and LCAD (1:500, Shanghai, China) diluted within 0.03% Triton X-100/PBS using 10% normal goat serum during the night at the temperature of 4 degrees. The following day, those sections were hatched using Alexa-Fluor 488 and Alexa-Fluor 568 secondary antibodies (1:250-1:500), and the nuclei were stained using Hoechst 33258 (Nacalai Tesque, Kyoto, Japan). Photographs were taken using a microscopic system connected with one digital camera. Besides, they were processed by means of Image and Photoshop.

Immunohistochemistry was performed as follows: fixation with 4% PFA for 20 min at room temperature; washing twice in PBS; permeabilization and blocking in blocking buffer (0.1% Triton X-100 and 3% FBS in PBS) for 1 h at room temperature; overnight incubation with primary antibodies diluted 1/500 in blocking solution; washing three times in PBS; incubation with Hoechst 33258 (Nacalai Tesque) and secondary antibody diluted 1/500 in blocking solution for 1 h in the dark at room temperature; and washing three times in PBS. Imaging was performed with a Leica AF6000 microscope. The primary antibody mouse anti-CDX2 (MU392A-UC, BioGenex) was used for immunostaining. CF488A donkey anti-mouse IgG (Biotium) secondary antibody was used to visualize signals. For the TUNEL assay, cells were stained with TMR Red using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions. For the EdU assay, EdU of the Click-iT EdU Imaging Kit (Invitrogen) was added to the ESC culture medium by exchanging half the medium and culturing for 4 h; the cells were then fixed with 4% PFA, permeabilized with 0.1% Triton X-100 in PBS, and stained with 1× Click-iT Reaction Buffer and Hoechst 33258.