Mouse anti tlr4 monoclonal igg

Cultured DRG neurons under different conditions were processed for double fluorescence labeling of MAP2 and NICD1 or TLR4. The double fluorescence labeling procedures were similar to our previous study^{34 (link)}. The first antibodies were mouse anti-NICD1 monoclonal IgG (1:500, Abcam, HK), mouse anti-TLR4 monoclonal IgG (1:500, Abcam, HK), mouse and chicken anti-MAP2 monoclonal IgG (1:500, Abcam, HK). The second antibodies were goat anti-mouse conjugated to Cy2 (1:100, Abcam, Cambridge, UK) and goat anti-chicken conjugated to Cy3 (1:200, Abcam, Cambridge, UK). Upon finishing fluorescence labeling, NICD1-IR and TLR4-IR neurons were counted in five visual fields of each coverslip. One visual field was in the center of the coverslip. The other four visual fields were just adjacent to, but not overlap with, the central visual field at the upper, lower, left, and right side, respectively. The MAP2-IR neurons in the same visual field were also counted as the total number of neurons. Then the percentage of NICD1-IR and TLR4-IR neurons could be obtained.

TLR4 and NICD1 protein levels in DRG from diabetic neuropathic pain animals and cultured DRG neurons under high glucose challenge were detected by Western blot assay. TNF-α protein levels in DRG from diabetic neuropathic pain animals were also detected by Western blot assay after Notch1 inhibitor DAPT or TLR4 inhibitor TAK treatment. The detailed Western blot assay procedures were as described in our previous study^{34 (link)}. The first antibodies used in this study were as follows: mouse anti-NICD1 monoclonal IgG (1:500, Abcam, HK), mouse anti-TLR4 monoclonal IgG (1:500, Abcam, HK), rabbit anti-TNF-α polyclonal IgG (1:1,000, Abcam, HK), and mouse anti-β-actin monoclonal IgG (1:1,000, Abcam, HK). The second antibodies were as follows: goat anti-rabbit IgG-HRP (1:3,000, Santa Cruz Biotechnology, Santa Cruz, CA) and goat anti-mouse IgG-HRP (1:3,000, Santa Cruz Biotechnology, Santa Cruz, CA). The relative levels of TLR4, NICD1, and TNF-α were normalized to control.