Ma5 32255

5 μm deparaffinized hippocampus tissue sections were rehydrated through a graded ethanol series and treated with a 3% H₂O₂ solution to quench endogenous peroxidases. The sections were immersed in a 0.1 M sodium citrate solution for antigen retrieval, followed by thorough rinsing with PBS (pH 7.4). The sections were blocked by incubation in 5% goat serum at 37°C for 30 min. After removal of the goat serum, the ROS probe (#88-5930-74, Invitrogen) and primary antibodies against IBA-1 (MABN92-AF647, Sigma-Aldrich), iNOS (#PA1-036, Invitrogen), arg-1 (#PA5-85267, Invitrogen), and NLRP3 (#MA5-32255, Invitrogen) were applied to the sections. The sections were counterstained with iNOS/IBA-1, arg-1/IBA-1, and NLRP3/IBA-1. Specifically, sections were incubated with the above probe and antibodies at 4°C for approximately 12 h. Then, the ROS probe and primary antibodies were removed, and the sections were incubated with fluorescence-labeled secondary antibody at 37°C for 30 min. DAPI staining (#62248, Thermo Scientific, USA) was used to label the cell nuclei. The stained sections were cover-slipped with an antifade mounting medium, and the fluorescent staining was visualized using a fluorescence microscope. Relative expression levels were analyzed using ImageJ software.

Deparaffinised spinal cord tissue sections were rehydrated and applied with 3% H₂O₂ solution and rinsed with PBS buffer. Antigen retrieval was done with applying 0.1 M sodium citrate solution. Then, sections were blocked with goat serum with a 30 min incubation at 37°C. Residual serum was removed by then. Primary antibodies against NeuN (#702022, Invitrogen, USA), iNOS (PA1-036, Invitrogen, USA), arg-1 (PA5-29645, Invitrogen, USA), IBA-1 (ab5076, Abcam, UK), NLRP3 (MA5-32255, Invitrogen, USA), and MK2 (ab131531, Abcam, UK) were applied to the sections followed by the overnight incubation at 4°C. After the removal of diluted primary antibody solutions, sections were rinsed with PBS buffer solution, ant incubated with fluorescent-labelled secondary antibody solution at 37°C for 30 min. DAPI staining solution was performed onto the sections for cell nucleus staining after thorough rinse with PBS buffer. Antifade reagent was applied to the sections after that to block the sections. Sections were then observed with a fluorescent microscope.