Goat anti rabbit alexa fluor 488

Brain vibratome sections were washed in 24-well plates with PBS, and sections were closed with 10 mL of 0.2% TritonX-100 and 150 μL of sheep serum protein for 2 h. Primary antibodies were incubated overnight at 4 °C. Primary antibodies included Iba−1 (1:100, AiFang, Changsha, China), GFAP (1:100, Proteintech, Wuhan, China), TNF−α (1:100, Proteintech, Wuhan, China), and IL−1β (1:100, ABclonal, Wuhan, China).
Sections were rinsed three times with PBS, incubated for 2 h at room temperature, and protected from light following the addition of secondary antibodies conjugated with Alexa Fluor 488 goat anti-rabbit (1:500, Beyotime, Shanghai, China) or goat anti-mouse cy3 (1:500, Beyotime, Shanghai, China). Sections were rinsed three times with PBS, attached to slides, dried in an oven at 37 °C for 5 min, and sealed with a sealer containing DAPI (SouthernBiotech, Birmingham, AL, USA). The images were observed and acquired using a Leica M205 fluorescence microscope. ImageJ (NIH, Bethesda, MD, USA) was used to count the percentage coverage of Iba-1 and GFAP in the area of positive staining in the field of view. The quantitative results concerning IL-1β and TNF-α were expressed as the cumulative fluorescence intensity in the field of view, and to intuitively observe the changes, the WTC group was used as a reference for normalized plotting.

For immunofluorescence staining of spinal cord tissue, the spinal cord tissue was dehydrated in 30 % sucrose solution for 3 days, embedded in OCT, and cut into 10 μm thick sections in transverse section. Cryofixed spinal cord sections were permeabilized and blocked in TBST with 1 % BSA at ambient temperature for 1 h. The sections were then incubated overnight at 4 °C with the following antibodies: Anti-NeuN (1:300, Abcam, USA), Anti-CD68 (1:200, Abcam, USA), Anti-iNOS (1:200, Abcam, USA), and Anti-Arg1 (1:200, Abcam, USA). To visualize the sections, Cy3-labeled goat anti-mouse (1:500, Beyotime, China) and Alexa Fluor 488 goat anti-rabbit (1:500, Beyotime, China) were used as secondary antibodies. Nuclear detection was performed by applying Fluoroshield Medium containing DAPI (Abcam, USA). Finally, confocal microscopy was used to capture images of the sections.

Cells seeded on glass slides were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% TritonX-100 for 10 min and then blocked with 5% BSA for 2 h at room temperature. Next, the cell slides were incubated with primary antibodies at 4 °C overnight. Then, the cell slides were washed with PBST three times and incubated with fluorescent-conjugated secondary antibodies-Goat anti-Rabbit Alexa Fluor 488 or Goat anti-Mouse Alexa Fluor 647 (Beyotime Institute of Biotechnology, Jiangsu, China) for 2 h at room temperature away from the light. Finally, the nucleus were stained with DAPI for 5 min. Fluorescence was visualized under laser confocal microscopy.