H324 500

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.

Immunohistochemistry (IHC) was performed on formalin-fixed paraffin-embedded (FFPE) patient sections with an automated staining system (Dako Link48) using TTF1 primary antibodies (ZSGB-Bio Cat: ZM-0270). After deparaffinization and rehydration in xylene and ethanol, antigen retrieval was performed in 1× EDTA retrieval solution (pH 8.0) (E-5134, Sigma) with heating. Inactivation of endogenous peroxidase was performed by adding enough drops of 3% hydrogen peroxide (H324-500, Fisher) to cover the whole section for 10 min and followed by primary antibody incubation at recommended dilution. After 60 min incubation at 37 °C, slides were washed twice with PBS, incubated with goat-anti-rabbit antibody (E046201, Dako) and then developed with DAB substrate (k3468, Dako) for 5–8 min depending on the antibodies. Slides were counterstained with hematoxylin (CTS-1090, Biotechnologies) and were scanned with brightfield pathology microscope at 20× magnification (Aperio CS2, Leica) and processed by ImageScope software (Leica). Information on all other chemicals and reagents used in the article can be found in Supplementary S-2.

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.