*5.7. Glial Cells Immunoreactivity Evaluated by Immunohistochemistry Assay*

At the 17th (peak of disease) and 28th (remission phase) days after immunization, animals were anesthetized using ketamine (75 mg/kg i.p.) and xylazine (10 mg/kg, i.p.) and were transcardially perfused with 0.9% saline followed by 4% of PFA solution. The spinal cord was collected, and tissues were included in PFA solution for 4 h to be post-fixed. After that, tissues were kept, for at least 48 h, in a 30% sucrose solution in a refrigerator (2–8 ◦C). After this period, the regions of the spinal cord corresponding to the L3-L6 were sectioned (30 μm) in a microtome, collected in an anti-freezing solution (500 mL of phosphate buffer 0.05 M, 300 mL of ethylene glycol, and 150 g of sucrose) and kept at −20 ◦C. After washing three times with phosphate buffer (PB 0.1M, pH 7.4), the material was incubated free-floating for 12–16 h with specific Iba-1 (ionized calciumbinding adaptor-1, 1:2000, Wako Chemicals, Richmond, VA, USA) and GFAP (glial fibrillary acidic protein, 1:1000, Sigma-Aldrich™, St. Louis, MO, USA) antibodies in PB containing 0.3% Triton X-100 and 5% normal donkey serum. After that, sections were washed in PB (3 times), incubated with the biotinylated secondary antibody (anti-rabbit, 1:200 in PB containing 0.3% Triton X-100) for 2 h, washed 3 times in PB, and incubated with the avidin complex -biotin peroxidase (ABC Elite, Vector) for 2 h. After the washing process, tissues were reacted in diaminobenzidine (DAB, Sigma Aldrich™, USA) and 0.01% hydrogen

peroxide in PB. The slices were mounted on gelatinized slides and kept at 37 ◦C for 48 h, dehydrated at room temperature, cleaned, and covered with a coverslip. Immunoreactivity quantification was performed in the dorsal horn of the spinal cord using ImageJ software (NIH/EUA).
