Anti ccr3

Immunohistochemical staining with anti-CD4 (1:500, Abcam, Cambridge, MA, USA), anti-COX-2 (COX-2; 1:1000, BioVision, Milpitas, CA, USA), anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), anti-NF-κB p65 (1:500, Abcam), and anti-MBP (1:500, Abcam), and immunofluorescence staining with polyclonal anti-BCL-2 (1:200; Santa Cruz, Dallas, TX, USA), anti-TNF-α (1:1000; ProSci Inc., San Diego, CA, USA), anti-activated BAX (1:200; Santa Cruz), anti-GAP43, and anti-CD68/ED1 (1:100; Santa Cruz) were performed as previously described (Cui et al., 2004 (link)). Double-immunostaining was performed using anti-CD4 (1:500, Abcam, Cambridge, MA, USA, green) and anti-CCR3 (1:500, Abcam, Cambridge, MA, USA) to identify Th2-polarized cells. Primary antibody omission controls were performed as negative controls.
Five sections from the motor cortex and bilateral anterior horns of the spinal cord for each animal were randomly selected and images were photographed under 20× magnification in three vision fields per section. The numbers of cells positive for COX-2-, NF-κB p65, GAP43, BAX, BCL-2 and caspase-3 were counted (cells positive for GAP43, BAX, and BCL-2 were counted via immunofluorescence staining and cells positive for COX-2-, NF-κB p65, and caspase-3 were counted using a counter-staining method).

Total proteins were extracted from eosinophils using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitors. The extracted proteins were quantified with a bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology). A total of 20 µg of protein was heated at 100°C for 5 min prior to loading, separated by 10% SDS–PAGE and subsequently transferred to a PVDF membrane (Merck KGaA). Following blocking for 1.5 h at room temperature with TBS containing 0.1% Tween and 5% fat-free powdered milk, the membranes were incubated overnight at 4°C with the primary antibodies anti-CCR3 (Cat no. Ab32512; 1:1,000; Abcam), anti-eotaxin-1 (Cat no. Ab25086; 1:1,000; Abcam), and GAPDH (Cat no. Ab181602; 1:1,000; Abcam). Following incubation with the primary antibody, the membranes were subsequently incubated at 4°C overnight with a secondary antibody (Cat no. 7074; 1:2,000; CST). The protein bands were detected using the enhanced chemiluminescence method (ECL; EMD Millipore). GAPDH served as the loading control for normalization of the protein levels.