2.7 Immunofluorescence staining and Image analysis
Immunofluorescence staining was performed as described previously(Ye, Cui et al., 2018). Anesthetized rats were perfused slowly with 200 ml of 0.01 M phosphate-buffered saline (PBS) and 200 mL of 4% paraformaldehyde. Whole brains were immediately removed, soaked in 4% paraformaldehyde at 4°C for 24 hours, and then successively transferred to 20% sucrose and 30% sucrose at 4°C until tissues were sunk. The brains were rapidly frozen in liquid optimal cutting temperature compound (Sakura Finetek, Cat# 4583, CA, USA) cooled with a mixture of solid carbon dioxide and ethanol. Serial coronal brain sections were cut in 20 μm thickness on a cryostat microtome (Leica Microsystems UK, Leica CM1950, Milton Keynes, UK) and stored at −20 °C in cryoprotectant solution (48% PBS, 30% ethylene glycol, 20% glycerol, 2% DMSO). To label the astrocytes and microglia, the sections were first placed in PBS (3×5 minutes) to wash out the cryoprotectant solution. Then, the antigen retrieval procedure was conducted in citrate buffer (Solarbio, Cat# C1032, China) for 5 minutes at 96°C. After cooling to room temperature, the sections were washed in PBS (3 × 5 minutes) again. Then, the sections were permeabilized with 0.3% Triton X-100 (Sigma‒Aldrich, Cat# T9284, MO, USA) for 20 minutes and blocked with 5% donkey serum at room temperature for 40 minutes. The sections were then incubated with primary antibodies against GFAP (1:500; Rabbit mAb, Cell Signaling Technology, Cat# 80788, RRID: AB_2799963) and Iba-1 (1:500; Rabbit mAb, Cell Signaling Technology, Cat# 17198, RRID: AB_2820254) overnight at 4°C respectively. After washing in PBS (3×5 minutes), sections were incubated with fluorescent-conjugated secondary antibodies (donkey anti-rabbit IgG Alexa Fluor 488, Abcam, Cat# ab150073, RRID: AB_2636877, 1:2000; donkey anti-rabbit IgG Alexa Fluor 647, Abcam, Cat# ab150075, RRID: AB_2752244, 1:2000) for 2 hours at room temperature. Finally, nuclei were subsequently stained with DAPI.
The single images from the PrL and IL for each mouse were scanned by a Nikon DS-Ri2 microscope camera (Nikon, Tokyo, Japan) and processed by Fiji software. All images of each section were acquired at 200× magnification. The morphological changes in microglia were evaluated following a previously published protocol (Young & Morrison, 2018). The number of branches and end-points were examined to investigate microglial morphological changes. These morphological parameters of microglia can reflect microglia complexity and distinguish the state of microglia. Sholl analysis protocol was based on those described earlier(Codeluppi, Chatterjee et al., 2021). In brief, bidimensional images were converted to 8-bit images and adjusted for brightness-contrast before thresholding to minimize background noise. Following these steps, the images were processed with the despeckle function and skeletozine. Then, the ending radius was depicted with a straight line. Last, we run the Sholl analysis with a set radius step of 3 μm.