Alexafluor 594 conjugated anti rabbit

Immunofluorescence staining was performed according to the protocols used for IHC analysis. The primary antibodies used were anti-IL-20 (7E) and anti-PD-L1 (Catalog: 17952-1-AP; Proteintech; 1:200 dilution). The secondary antibodies used were AlexaFluor 488-conjugated anti-human (Catalog: 109-545-003; Jackson ImmunoResearch; 1:200 dilution) and AlexaFluor 594-conjugated anti-rabbit (Catalog: 111-585-003; Jackson ImmunoResearch; 1:200 dilution) antibodies.

Primary antibodies used for immunostaining were anti-TRF1 (Abcam, ab192629, 1:100), anti-BubR1 (Abcam, ab28193, 1:100), anti-Zw10 (Abcam, ab21582, 1:100), anti-acetylated-α-tubulin (Sigma Aldrich, T7451, 1:500; Abcam, ab179484, 1:500), anti-centromere (Antibodies Incorporated, 15-234, 1:100), anti-CENP-A (Cell Signaling, #2048, 1:100), anti-Survivin (Cell Signaling, #2808, 1:100), anti-Hec1 (Santa Cruz Biotechnology, sc-515550, 1:100), anti-H3T3ph (Upstate, 07-424, 1:100), anti-SMC3 (Abcam, ab128919, 1:100), anti-SMC4 (Novus Biologicals, NBP1-86635, 1:100), and anti-TRF2 (Abcam, ab13579, 1:100). Secondary antibodies were Alexa Fluor 488-conjugated anti-mouse (Jackson ImmunoResearch, 115-545-144 1:500), Alexa Fluor 594-conjugated anti-mouse (Jackson ImmunoResearch, 111-585-146, 1:500), Alexa Fluor 488-conjugated anti-rabbit (Jackson ImmunoResearch, 115-545-144 1:500), Alexa Fluor 594-conjugated anti-rabbit (Jackson ImmunoResearch, 111-585-144, 1:500), and Alexa Fluor 488-conjugated anti-sheep antibodies (Abcam, ab150177, 1:500).

AMs from the BALF of the vehicle or OVA-challenged mice were isolated as described above and seeded in 24-well plates containing a coverslip. After adherence, the cells were washed by PBS and fixed with 10% formaldehyde. After permeabilizing in PBS containing 0.5% Triton X-100 and blocked with 5% donkey serum, the cells were incubated with F4/80 (Santa Cruz) and Irg1 (Abcam, Boston, MA, USA) antibodies, and F4/80 staining was used to identify the macrophage. The second antibody was the Alexa Fluor 594^®-conjugated anti rabbit or Alexa Fluor 488^®-conjugated anti mouse IgG (Jackson ImmunoResearch Lab, Inc. West Grove, PA, USA). Then, the coverslips were counterstained with DAPI and observed with confocal microscopy (Zeiss LSM 800, Carl Zeiss AG, Oberkochen, Germany, 63 × 1.4 oil).