Mab3651

Table 2 shows the series of antibodies used in this study. The antibody against type IV collagen was used to demarcate the basal lamina (BL) of neurons, blood vessels, and epithelium. We used a polyclonal antibody (1:25, goat ab, AB769, Millipore). For residents’ macrophages, we used antibody against IBA1 (polyclonal, 1:100, rabbit, PA527436 from Invitrogen). Specificity was proven by IBA1 antibody blotting (36 (link)). Fractalkine antibody was a monoclonal antibody (1:100, mouse, MAB3651, R&D Systems). This antibody specificity antibody was verified in western blotting experiment (37 (link)). Information about the other primary and secondary antibodies is shown in Table 2.

Proteins from plasma membrane fractions were incubated for 2 h at 4 °C at a final concentration of 2 mg/ml in 50 mM phosphate buffer pH 8.0, 200 mM NaCl, 1X protease inhibitor cocktail, 10% glycerol and with 5 Critical Micellar Concentration of CALX173ACE (CALIXAR). CALX173ACE solubilized CX3CL1 was loaded into magnetic beads previously crosslinked to polyclonal anti-CX3CL1 antibody using an IP kit (Pierce, Thermo Fisher Scientific). Retained CX3CL1 was eluted by pH shock (basic) followed by a neutralization step. Non-denaturated proteins were separated by native-PAGE on a 4–15% acrylamide gel (4–15% Mini-PROTEAN TGX Stain-Free™ Gel, Bio-Rad) using 25 mM imidazole pH 8.0 as anode buffer and 50 mM Tricine, 7.5 mM imidazole, 0.05% deoxycholate and 0.01% DDM as cathode buffer). Clear Native PAGE gels ran for 90 min at 200 V and 4 °C. Proteins were then immobilized by electro-transfer to PVDF membrane. The immunodetection of CX3CL1 was performed by using the SNAP i.d. system (Millipore) with primary antibody (R&D system, MAB3651) against CX3CL1 (1/500 dilution) and revealed using a mouse HRP secondary antibody (Santa Cruz, 3/10,000 dilution).