Alexa fluor 488 conjugated avidin

For lnc-EPAV RNA FISH assay, BMDMs cultured on poly-l-lysine-coated coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, washed, and stored with 70% ethanol at −20°C. Endogenous biotin signal was blocked by using an endogenous biotin blocking kit (Thermo Fisher, USA). BMDMs were incubated with biotin-labeled lnc-EPAV probe at 50°C overnight. The cells were then incubated with Alexa Fluor 488-conjugated avidin (Thermo Fisher, USA) at room temperature for 4 h. For immunofluorescence analysis, cells were sequentially fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% bovine serum albumin (BSA), and incubated with primary antibody (mouse anti-SFPQ; Sigma, USA) followed by Alexa Fluor 546-conjugated goat anti-mouse secondary antibody (Thermo Fisher, USA). Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Images were acquired using a Carl Zeiss LSM710 microscope (objective, 40×).

Fluorescent in situ hybridization was performed according to the protocol of Cremer et al. (2008) [37 (link)] on 30 μm-thick brain cryosections of the 4% paraformaldehyde-perfused, 8 kainate-treated, and 4 control animals, as well as on 3 independent primary hippocampal neuronal cultures fixed with 4% paraformaldehyde at DIV 14 (day in vitro). As templates for Bdnf FISH probes, CH230-449H21 BAC obtained from Children’s Hospital Oakland Research Institute were used. Probes were verified on rat metaphase spreads. The probes were labeled using the standard nick-translation procedure. Biotinylated probes were detected using Alexa Fluor 488- conjugated avidin (Invitrogen), followed by FITC-conjugated rabbit anti-avidin antibody (Sigma-Aldrich).