Alexa fluor donkey anti rabbit igg

Ganglion cells and their fibers were observed using an antibody against neurofilaments (Ding et al., 2011 (link); Ding et al., 2013 (link); Dong et al., 2018 (link)). Whole-mount preparations were incubated overnight at 4 °C with an anti-neurofilament antibody (rabbit anti-Neurofilament 200 antibody, N4142, Sigma-Aldrich, St. Louis, MO, USA) diluted in 1% Triton X-100 and 5% donkey serum in 0.1 M PBS (1:100). After incubation with the primary antibody, the tissues were rinsed 3 times with 10 mM PBS and incubated for 2 hours at room temperature with a secondary antibody (1:100 Alexa Fluor® donkey anti-rabbit IgG, Life Technologies, Grand Island, NY, USA) in 10 mM PBS. The specificity of the primary antibody was confirmed in our previous publications (Ding et al., 2011 (link); Ding et al., 2013 (link); Dong et al., 2018 (link)).

To determine the location of macrophages, we used spiral ganglion neurons and their peripheral and central processes as a landmark for identifying macrophages in neural regions. Ganglion cells and their fibers were observed using the immunoreactivity of tubulin, the dimeric structural protein of microtubules, using a method that has been described in our previous publications (Ding et al., 2011 (link); Ding et al., 2013 (link)). After dissection, whole-mount preparations were incubated overnight at 4°C with an anti-Tubulin antibody (Rabbit anti-Tubulin, ab59680, Abcam Inc., Cambridge, MA, USA) diluted in 1% Triton X-100 and 5% donkey serum in 0.1 M PBS (1:100). The tissues were counterstained with the CD45 or F4/80 primary antibody as described above. After incubation with the primary antibodies, the tissues were rinsed 3 times with distilled water and incubated for 2 hours at room temperature with secondary antibodies (Alexa Fluor® donkey anti-rabbit IgG and Alexa Fluor® donkey anti-goat IgG, 1:100, A10042, Life Technologies, Grand Island, NY, USA) in 1% Triton X-100 and 5% donkey serum in 0.1 M PBS. The specificity of the primary antibody was confirmed in our previous publications (Ding et al., 2011 (link); Ding et al., 2013 (link)).