Anti sod1

Six samples from SCV in each group were deparaffinized in xylene and rehydrated by a series of ethanol concentrations from 100% to 75%. Sections were subsequently immersed in 3% hydrogen peroxide solution to inhibit endogenous peroxidase activities and incubated in 10% normal goat serum. Finally, sections were incubated at 4°C overnight against the following primary antibodies: anti-Cadherin 5 (1:100, Boster Biological Technology, China), anti-CD34 (1:200, Abcam, UK), anti-VEGF (1:200, Abcam), anti-CTSD (1:200, Proteintech, USA), anti-SOD1 (1:200, Proteintech), and anti-C-CASP3 [1:200, CST (Cell Signaling Technologies, USA)]. These sections were then incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, USA) and counterstained with hematoxylin. Each specimen was imaged at a magnification of ×200 using a DP2-BSW image-acquisition system (Olympus Corp), and absorption values quantified using the Image-Pro Plus software (Media Cybernetics, MD, USA) to assess expressions of Cadherin 5, VEGF, CTSD, SOD1, and C-CASP3 and the number of CD34 positive microvessels.

Spinal cord section homogenates were resolved in pre-cast 4–20% SDS/PAGE (Bio-Rad, mini-PROTEAN TGX™ gels; Cat#456–1095) under the reducing conditions. The proteins were transferred onto a transfer membrane (Millipore, Immobilon® PVDF membrane, Cat#IPFL00010) and probed with anti-Calcineurin_α, (Cat#C1956; Sigma-Aldrich, St Louis, MO, USA; monoclonal antibody) anti-Calcineurin_β, (Cat#C0581; Sigma-Aldrich, St Louis, MO, USA; monoclonal antibody), anti-SOD1 (Cat#100269-1-AP; Protein Tech, USA, Rabbit polyclonal antibody), and anti-TDP-43 (Cat#10782-2-AP; Protein Tech, USA) antibodies. Odyssey/LI-COR detection system (LI-COR Biosciences, Nebraska, USA) was used for analyzing the protein bands (Image Studio Ver.3.1).

The processed samples mentioned above were then deparaffinized in xylene, and different concentrations from 100% to 75% of ethanol baths were applied for rehydration. Afterward, the rehydrated specimens were immersed in 3% (v/v) hydrogen peroxide to block endogenous peroxide activities and kept in 10.2 mM of sodium citrate for antigen repair at 95°C for 20 min. Finally, samples were incubated overnight at 4°C with the following required primary antibodies, including anti-Cadherin 5 (1:100, Affinity, Cincinnati, OH, USA), anti-CD34 (1:100, Abcam, Cambridge, UK), anti-VEGF (1:300, Abcam), cleaved caspase-3 (1:200, Cell Signaling Technology, Danvers, MA, USA), anti-SOD1 (1:100, ProteinTech, Chicago, IL, USA), or anti-CTSD (1:100, ABclonal, Woburn, MA, USA), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody, and stained again with hematoxylin. The DP2-TWAN image-acquisition system (Nikon, Tokyo, Japan) was performed to acquire images of the tissue sections on the light microscopy at ×200 magnification. Images were analyzed to quantify for Cadherin 5, CD34, VEGF, C-CASP3, SOD1, and CTSD expression levels; and the amount of CD34-positive microvessels was enumerated. Assessments were acquired from six random visual fields in three tissue sections.