Brightvision plus kit

For double‐labeling after ISH, slides were incubated for 1 h at room temperature with the following primary antibodies: mouse anti‐glial fibrillary acidic protein (GFAP; 1:4000, Sigma‐Aldrich, St. Louis, MO, USA), mouse anti‐NeuN (1:2000, MAB377, Chemicon, Temecula, CA, USA), rabbit anti‐ionized calcium binding adaptor molecule 1 (Iba1; 1:2000, Wako Chemicals, Neuss, Germany), mouse anti‐human leukocyte antigen (HLA‐DR/DP/DQ; 1:100, clone CR3/43 Agilent, Santa Clara, CA, USA), rabbit anti‐transmembrane protein 119 (TMEM119; 1:500, #HPA051870, Sigma‐Aldrich, St. Louis, MO, USA), mouse anti‐CD68 (1:200, clone KP1, Dako, Glostrup, Denmark) or rabbit anti‐CD8 (1:200, #7103, Dako, Glostrup, Denmark). Secondary horseradish peroxidase (HRP)‐conjugated antibodies (Brightvision plus kit, ImmunoLogic, Duiven, the Netherlands) were used. The visualization of the antibody‐antigen binding was done using 3‐amino‐9‐ethylcarbazole (AEC; Sigma‐Aldrich, St. Louis, MO, USA).

In situ hybridization was performed on a subset of 7-d post-TBI sections as described above. After in situ hybridization, the sections were washed three times with phosphate-buffered saline (PBS) and incubated with primary antibody against glial fibrillary acidic protein (astrocytes, 1:2000, G-3893, Clone G-A-5, Sigma-Aldrich, Munich, Germany), Iba1 (microglia, 1:200, 019-19741, WAKO, Tokyo, Japan), or NeuN (neuronal nuclei, 1:2000, MAB377, clone A60, Chemicon, Temecula, CA, USA) in room temperature for 60 min. The sections used for NeuN detection were then washed three times with PBS and blocked with post-antibody blocking solution (BrightVision plus Kit, Immunologic, Duiven, The Netherlands) at room temperature for 15 min. All sections were then washed three times with PBS. Poly-HRP-GAMs/Rb IgG was added to the sections and the sections were incubated at room temperature for 30 min. To detect the peroxidase activity, sections were incubated with aminoethylcarbazole (in 0.05 M saline buffer, pH 4.9 with 0.01% H₂O₂) for 8 min in the dark. To stop the reaction, the sections were first washed in deionized water then in tap water. The sections were dehydrated by dipping them into 70% ethanol for 1 min, twice into absolute ethanol for 1 min, and three times into xylene for 1 min. Finally, the sections were coverslipped using glycerol gelatin.