Ab125212

Hepatic specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Liver sections (4-µm thick) were cut serially and stained with an H&E staining kit based on the instructions. Immunohistochemistry was performed on the sections with anti-S100A9 (73425, CST, Danvers, MA, USA) or anti-CD68 (ab125212, Abcam, Cambridge, MA, USA) antibodies. Immunofluorescent staining was conducted with anti-S100A9 (73425, CST) and anti-F4/80 (6640, CST) antibodies to assess the localization of S100A9 in macrophages. The histological score of H&E tissue staining was determined based on four grades according to a previous study [39 (link)].
To evaluate liver cell apoptosis, tissue cryosections at 6 µm thickness were stained by using a TUNEL Apoptosis Detection kit (Roche, Basle, Switzerland) based on previously described protocols [40 (link)]. The ratio of TUNEL-positive cells to DAPI-positive nuclei was calculated and presented as the percentage of apoptotic cells for each group. For superoxide analysis, cryosections were stained with 1 µmol/L dihydroethidine (DHE, Sigma, St. Louis, MO, USA) in a PBS buffer (pH 7.4) for 30 min at 37 °C, as described previously [41 (link)]. Fluorescence images were obtained from 6–8 random fields for each sample with a Nikon microscope (Nikon, Tokyo, Japan).

The paraffin-embedded sections (4 μm) of the spinal cords were mounted on glass slides. IHC analysis of the sections was performed using an automated slide preparation system (Benchmark XT; Ventana Medical Systems Inc., Tucson, AZ, USA). Deparaffinization, epitope retrieval, and immunostaining were performed using cell conditioning solutions and the BMK ultraVIEW diaminobenzidine detection system (Ventana Medical Systems Inc., Tucson, AZ), following the manufacturer’s instructions. The tissue sections were stained with anti-CD3 (#ab16669, Abcam), anti-CD68 (#ab125212, Abcam), and anti-PAX5 (#ab109443, Abcam) primary antibodies. The positive signals were amplified using ultraVIEW copper. The sections were counterstained with hematoxylin and bluing reagent. The number of CD3-, CD68-, and PAX5-immunoreactive cells in the white matter and pia mater of the spinal cord was examined using a light microscope and normalized to the total area of white matter and pia mater.

Antibodies for CD3 (Ab5690), CD4 (Ab183685), CD68 (Ab125212), alpha fetoprotein (α-AFP) (Ab46799), and Glypican-3 (GPC-3) (Ab66596) were purchased from Abcam Inc. (Cambridge, MA). Antibodies for SV40 TAg (v-300) (sc-20800) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Antibodies for CD8a (14-0808) and F4/80 (14-4801-81) were purchased from Affymetric eBioscience, (San Diego, CA).