Alexa fluor labelled secondary antibody

The mice were euthanized, and their eyes were enucleated and fixed with freshly prepared 4% paraformaldehyde for 0.5 hr. Retinae were dissected, permeabilized in 0.5% Triton X-100 for 15 minutes, blocked in 5% BSA for 1 hr at room temperature (RT), and incubated overnight at 4 °C with a rabbit Ab against Iba-1 (ionized calcium-binding adaptor molecule 1, 1:200; Wako, Osaka, Japan), which was followed by an incubation with an Alexa Fluor labelled secondary antibody (Cell Signaling Technology, Beverly, MA) for 1 hr at RT after 3 washes with PBS. Next, the retinae were washed again and mounted on microscope slides. The retinae were examined under confocal microscopy (LSM710; Carl Zeiss). The number of Iba1 positive cell was quantified.

NRCMs cultured on coverslips were fixed using 4% paraformaldehyde, washed with warm PBS, permeabilized with 0.3% Triton X-100 at room temperature for 10 min and subsequently incubated with 10% goat serum (BOSTER, Wuhan, China) at room temperature for 1 h. Afterwards, cells were incubated with primary antibody of PARP1 (diluted 1:50), PAR (diluted 1:50) and C/EBPβ (diluted 1:50) at 4 °C overnight, and further incubated with Alexa Fluor-labelled secondary antibody (diluted 1:200, Cell Signaling Technology, Danvers, MA, USA). Finally, the coverslips were mounted with DAPI (Cell Signaling Technology, USA). Confocal microscope (Zeiss, Germany) was used to detect the coverslips.