Canada balsam

The following antibodies were used: polyclonal rabbit anti-NOX4 (ab3517, Abcam, Cambridge, UK), polyclonal rabbit anti-4-HNE antibody (ab3517, Abcam, Cambridge, UK), polyclonal rabbit anti-Malondialdehyde (MDA) antibody (ab27642, Abcam, Cambridge, UK), monoclonal mouse anti-OXPHOS complex antibody (ab110413, Abcam, Cambridge, UK), monoclonal mouse anti-GFAP (#3670, Cell signaling technology, Danvers, MA, USA), polyclonal rabbit anti-Tomm20 antibody (sc-17764, Santa Cruz Biotechnology, Dallas, TX, USA), polyclonal rabbit anti-NRF2 antibody (#12721, Cell signaling technology, Danvers, MA, USA), polyclonal rabbit Histone H3 antibody (#9715, Cell signaling technology, Danvers, MA, USA) and monoclonal mouse anti-β-actin (A5316, Sigma-Aldrich, St. Louis, MO, USA). Fluoroshield™ with DAPI (F6057, Sigma-Aldrich, St. Louis, MO, USA) was used for nuclear staining and mounting. Sections were mounted onto gelatin-coated slides with Canada Balsam (Wako, Tokyo, Japan) following dehydration.

The cavity volume was measured and calculated as described previously (Nakano et al., 2013 (link)). In brief, three rats each from CPEC-transplanted and PBS-injected groups were used at 5 w-TP. Horizontal cryostat sections of the spinal cord were stained by hematoxylin and eosin (HE) on the slide glass. After staining, the sections were embedded in Canada balsam (Wako, Osaka), and used for measurement of the volume of cavities. The border of cavities was traced to measure the areas of the cavities using the Image Filing System (Flovel, Tokyo, Japan) equipped on a microscope (Olympus, CKX41, Tokyo, Japan). The areas of the cavities were measured in every third section. Since the sections were ca. 10 μm thick, the total volume of the cavities was calculated by multiplying the average area of the cavities by the depth of the sections examined. The relative volume of the cavities was obtained by dividing the values of the total cavity volume by those of the whole spinal cord volume at the corresponding level.

The following antibodies were used: monoclonal rabbit anti-TXNIP (#14715, Cell signaling technology, Danvers, MA, USA), monoclonal mouse anti-TXNIP (NBP1-54578, Novus Biologicals, Centennial, CO, USA), monoclonal mouse anti-OXPHOS complex antibody (ab110413, Abcam, Cambridge, UK), monoclonal rabbit anti-cleaved caspase 3 (#9661, Cell signaling technology), monoclonal mouse anti-GFAP (#3670, Cell signaling technology), monoclonal goat anti-GFAP (ab302644, Abcam), polyclonal rabbit anti-Tomm20 antibody (sc-17764, Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal rabbit anti–NF–κB p65 (D14E12) (#8242, Cell signaling technology), monoclonal rabbit anti-phospho–NF–κB p65 (Ser536) (93H1) (#3033, Cell signaling technology), monoclonal rabbit anti-NRF2 antibody (#12721, Cell signaling technology), polyclonal rabbit Histone H3 antibody (#9715, Cell signaling technology), and monoclonal mouse anti-β-actin (A5316, Sigma-Aldrich, St. Louis, MO, USA). Fluoroshield™ with DAPI (F6057, Sigma-Aldrich) was used for nuclear staining and mounting. Sections were mounted onto gelatin-coated slides with Canada Balsam (Wako, Tokyo, Japan) following dehydration.