Alexa fluor594 goat anti

Slides were incubated with the following primary antibodies at 4°C overnight (8 hours): rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1; 1:500; Abcam, Cambridge, UK, Cat# ab178846; RRID: AB_2636859), as Iba-1 is up-regulated in microglia following nerve injury (Wu et al., 2022); mouse anti-glial fibrillary acidic protein (GFAP; 1:300; Cell Signaling Technology, Danvers, MA, USA, Cat# 3670S; RRID: AB_561049), as GFAP is expressed in the central nervous system in astrocytes (Kisucká et al., 2021); and rabbit anti-neurofilament-200 (NF200; 1:2000; Cell Signaling Technology, Cat# 2836S; RRID: AB_10694081), as NF200 is expressed in the central nervous system in neurofilaments (Xu et al., 2015). Next, the slides were incubated with fluorescent Alexa Fluor488 goat anti-mouse (1:1000; Invitrogen, Carlsbad, CA, USA, Cat# A32723TR; RRID: AB_2866489) and Alexa Fluor594 goat anti-rabbit secondary antibodies (1:1000, Invitrogen, Cat# A32740; RRID: AB_2762824) for 60 minutes at 37°C. An inverted fluorescence microscope (Leica DM6000, Wetzlar, Germany) was used to capture images and conduct further analysis. To determine the total area of neurofilament protein, the NF200-positive area in spinal cord was calculated. Iba-1 and GFAP-positive areas were observed to assess aggregation of microglia and astrocytes, respectively (Zeng et al., 2019).