Alexa fluor 594 conjugated donkey anti rabbit igg

Monocytes on the cytospin slides were fixed in 10% neutral buffered formalin for 20 min at room temperature. After washing with 1xPBS for three times (5 min each), the nonspecific protein binding was blocked by incubating the cells with 3% BSA, 5% normal donkey serum, and 0.3% Triton X-100 for 30 min at room temperature. Then cells were incubated with primary antibodies CD11b (1:50, cat. no. PA1943, Boster Biological, Pleasanton, CA, USA) with Ly-6G (1:50, cat. no. 551459, BD, San Jose, CA, USA) or Alexa Fluor 488-conjugated F4/80 (1:20, cat. no. MF48020, Invitrogen, Carlsbad, CA, USA). CD11b is a marker of monocytes, while Ly-6G is a marker of neutrophils (Geissmann et al., 2003 (link); Swirski et al., 2006 (link)). Activated macrophages exhibited F4/80 positive, while monocytes showed F4/80 low (Geissmann et al., 2003 (link); Swirski et al., 2006 (link)). After incubation with primary antibodies for 2 h at room temperature and washed with 1xPBS three times, the cells were incubated with Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:500, Abcam, Cambridge, MA, USA) with/without Alexa Fluor 488-conjugated donkey anti-rat IgG (1:500, Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h. After washing, the slides were mounted with Prolong Gold Antifade Reagent with DAPI (Invitrogen, Carlsbad, CA, USA) and examined by fluorescence microscopy.