Hoechst pi

The viability of primary cortical neurons was estimated by Hoechst 33258 and propidium iodide (Hoechst/PI; Sigma-Adrich) co-staining and visualized using a fluorescence microscope Olympus IX71, as previously described (Avdoshina et al., 2016a (link)). Hoechst/PI-positive cells were then counted using ImageJ (National Institutes of Health, Bethesda, MD, United States) and expressed as a percentage of the total number of neurons.

NC siRNA (5′-UUCUCCGAACGUGUCACGU-3′; Genepharma, China), FOXQ1 siRNA (5′-CGCGGACUUUGCACUUUGA-3′; Genepharma, China), PTGS2 siRNA (5′-UCAAGUGUUGCACAUAAUCDTDT-3′; Genepharma, China), FOXQ1 siRNA (5′-CGCGGACUUUGCACUUUGA-3′; Genepharma, China) combining PTGS2 siRNA (5′-UCAAGUGUUGCACAUAAUCDTDT-3′; Genepharma, China), CDK5 siRNA (5′-GUCGAUGACCAGUUGAAGATT-3′; Genepharma, China), and FOXQ1 siRNA (5′-CGCGGACUUUGCACUUUGA-3′; Genepharma, China) combining CDK5 siRNA (5′-GUCGAUGACCAGUUGAAGATT-3′; Genepharma, China) were transfected into two cellular AD models (primary neuron AD model and PC-12 cellular AD model) using Lipofectamine™ LTX Reagent with PLUS™ Reagent (Invitrogen, USA). Then the transfected cells were divided into NC group, Si-FOXQ1 group, Si-PTGS2 group, Si-FOXQ1&Si-PTGS2 group, Si-CDK5 group, and Si-FOXQ1&Si-CDK5 group. At 48 h, the cell apoptosis rate was evaluated by Hoechst/PI (Sigma, USA) in accordance with the method in the previous study (Ma et al., 2019 (link)); neurite outgrowth was observed under ×200 magnification with a microscope (Olympus, Japan), and then the TNF-α, IL-1β, and IL-6 in supernatant were measured by ELISA, and the expressions of FOXQ1, PTGS2, and CDK5 in each group were detected by Western blot.

Chick embryos were harvested after a given time incubation and fixed in 4% PFA overnight at 4 °C. Whole-mount embryo immunostaining was performed using the following antibody: MF-20 (1:500, DSHB, USA). Briefly, the fixed embryos were then incubated with this primary antibody at 4 °C overnight on a shaker. Following extensive washing, the embryos were incubated with anti-rabbit IgG conjugated to Alexa Fluor 488 overnight at 4 °C on a rocker. For F-actin detection, the cultured cells were stained using phalloidin-Alexa-Fluor 488 (1:200, Invitrogen, USA) at room temperature for 2 h. All the embryos were later counterstained with 4',6-diamidino-2-phenylindole (DAPI; 1:1000, Invitrogen, USA) at room temperature for 1 h. For Hoechst (1:1000, Sigma, USA)/propidium iodide (PI, 1:1000, Sigma, USA) staining, the cells were cultured and washed twice with cold phosphate-buffered saline (PBS), and then incubated with Hoechst/PI for 45 min at 37 °C in the dark.