4 streptavidin hrp label

Sections were cut, deparaffinised, and rehydrated as described above. Antigen retrieval was performed for 30 s at 123°C in Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) using a pressure cooker followed by a cool-down period of 20 min. Reduction of peroxidases was accomplished by incubating for 8 min in Peroxidazed 1 (BioCare Medical, Concord, CA). Slides were sequentially blocked in Avidin-Biotin Blocking Kit (BioCare Medical), Background Sniper (BioCare Medical) and Rodent Block M (BioCare Medical) for 15 min, respectively. Biotinylated Hyaluronic Acid Binding Protein (Millipore, Billerica, MA) was added at a 1:800 dilution and incubated for 1 h at room temperature followed by a 10 min incubation with 4+ Streptavidin HRP Label (BioCare Medical). Staining was performed with Betazoid DABKit (BioCare Medical) for 5 min followed by counterstaining with CAT Hematoxylin (BioCare Medical) for 1 min.

To evaluate the axonal changes in nerve root lesions in the lumbar spinal cord segment, we selected a CC002 mouse and a CC023 mouse for neurofilament immunohistochemistry. No antigen retrieval was performed. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide (Fisher H324-500) for 5 minutes. Universal Blocking Reagent 10X (Power Block) (BioGenex HK085-5K) was applied for 5 minutes. Sections were then incubated for 60 minutes with the anti-neurofilament, clone NE-14 (BioGenex MU073-UC [1:1000]) followed by a 10 minute incubation with biotinylated anti-mouse (Vector BA-9200, 1:100). Detection was performed with 4+ Streptavidin HRP Label (Bio Care Medical, Ap604H) for 10 minutes and Beazoid DAB Chromagen Kit (Bio Care Medical, BDB2004L) for 12 minutes.

One sham mouse and three infected mice for each strain were selected at 4 dpi and 14 dpi timepoints for Iba-1 immunohistochemistry. Iba-1 is expressed by microglia and macrophages and is involved in early phagocytosis; it is commonly used as a pan-macrophage marker [13 (link),53 (link)]. The immunohistochemistry protocol was optimized and standardized using an autostainer. The following protocol was used: Antigen retrieval was performed with concentrated Antigen Retrieval Citra Solution (BioGenex HK986-9K, Fremont, CA, USA) for 15 min at 110 °C. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide (Fisher H324-500, Hampton, NH, USA) for 5 min. Universal Blocking Reagent 10X (Power Block) (BioGenex HK085-5K, Fremont, CA, USA) was applied for 5 min. Sections were then incubated for 60 min with rabbit antibody against Iba-1 (019-19741, Wako Chemicals, Richmond, VA, USA [1:8000]) followed by a 10-min incubation with biotinylated Goat Anti-Rabbit (Vector BA-1000, 1:100). Detection was performed with 4+ Streptavidin HRP Label (Bio Care Medical, Ap604H, Pacheco, CA, USA) for 10 min and Beazoid DBA Chromagen Kit (Bio Care Medical, BDB2004L, Pacheco, CA, USA) for 12 min.