Alexa fluor 488 conjugated goat anti mouse secondary antibody

The methods of material fixation and slide preparation are given in Cheng (2013) (link). After removing the coverslip, the slides were marked by a stain circle pen and incubated in washing buffer I (1×PBS with 1% (v/v) Triton X-100) for an hour at room temperature. Then slides were incubated with the primary antibodies, including anti-γH2AX (raised in rabbit; Miao et al., 2013 (link)), OsREC8 (raised in both rabbit and mouse; Shao et al., 2011 (link)), OsMER11 (raised in mouse; Ji et al., 2013 (link)), OsCOM1 (raised in mouse; Ji et al., 2012 (link)), OsDMC1 (raised in mouse; Wang et al., 2016 (link)), OsMER3 (raised in mouse; Wang et al., 2009 (link)), OsPAIR2 (raised in mouse; Wang et al., 2009 (link)) or anti-OsZEP1 (raised in mouse; Wang et al., 2010 (link)) antibody solution (diluted 1:200 in blocking buffer: 1×PBS, 1 mM EDTA, 0.1% Tween 20, 5% BSA), at 4 ^oC overnight. After three rounds of washing in washing buffer II (1×PBS with 0.1% (v/v) Tween 20), Alexa Fluor 488-conjugated goat anti-mouse secondary antibody or Alexa Fluor 555-conjugated donkey anti-rabbit secondary antibody (Beyotime, Shanghai, China) was added to the slides. The chromosomes were counterstained with DAPI (10 mg mL^–1) in an anti-fade solution (Vector Laboratories).

Mφs on the climbing piece in 24-well culture plates were treated with or without bacterium infection or treated with relevant CM for 48 h. Then the cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 0.01% Triton X-100 for 10 min. After being washed with PBS, the cells were blocked with 10% goat serum for 30 min at room temperature and incubated with anti-CD86 monoclonal antibodies (Santa Cruz, USA) and anti-CD206 antibodies (Proteintech, China) at 4°C overnight. The next day, the cells were rinsed with PBS for clearing the primary antibody, and then incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Beyotime, China) or Cy3-conjugated goat anti-rabbit secondary antibody (Beyotime, China) at room temperature for 1 h in the dark, and then washed with PBS, and counterstained the nucleus with DAPI for 10 min. After washing again with PBS and mounting with antifade polyvinylpyrrolidone mounting medium (Beyotime, China), the fluorescent images were observed using confocal microscope (Lecia, Germany).