Alexa fluor conjugated secondary ab

Primary and secondary Abs used for immunolabeling: mouse anti-Ki67 Ab (Dako), rat anti-BrdU (Abcam), mouse anti-Muc2 and rabbit anti-chromogranin A (Santa Cruz), rabbit anti–caspase-3 (Cell Signaling); rabbit anti–E-cadherin (BD Bioscience); rat or rabbit F4/80, rat anti-7/4 (Abcam); rat or rabbit anti-Ly6C, Ly6G, and Gr-1 (BD Pharmingen); rabbit anti-CD11b; and rat IgG and rabbit IgG (Abcam). Immmunolabeling was visualized using an appropriate combination of species-specific Alexa Fluor–conjugated secondary Abs (488, 568, and 647 nm) raised in mouse, donkey, or goat (Invitrogen).

Cells grown on 14-mm coverslips (Fisher Scientific) were fixed with 4% formaldehyde in 1X PBS at room temperature for 15 min (in fume hood), rinsed three times in PBS and permeabilized or not with ice-cold 100% methanol for 10 min at -20°C. After 5 min of wash in PBS, nonspecific binding was blocked by 30 min incubation with blocking buffer (1X PBS-5% normal goat serum with or without 0.3% Triton X-100) at room temperature. Coverslips were then incubated overnight at 4°C in Ab dilution buffer (1X PBS-1% BSA with or without 0.3% Triton X-100) with the primary Ab. The following primary Abs were used: rabbit polyclonal anti-IL-22R1 Ab (10 μg/mL; Abcam), mouse monoclonal anti-IL-10R2 Ab (5 μg/mL; R&D Systems), rabbit monoclonal anti-phospho-STAT3 (Tyr705) (1:100; Cell Signaling Technology) and mouse monoclonal anti-STAT3 Ab (1:1600; Cell Signaling Technology). Cells were washed 3 times in PBS, and incubated for 2 h at room temperature (in dark) with 488 nm Alexa Fluor-conjugated secondary Abs (Invitrogen) diluted 1:1000 in Ab dilution buffer. After 3 washes in PBS, nuclei were stained for 5 min with DAPI (Sigma). After intensive washes, coverslips were inverted on slides and mounted with Dako Fluorescent Mounting Medium. Isotype controls were cells incubated with irrelevant normal rabbit or mouse IgG (Sigma). Pictures were taken using a confocal microscope (Carl Zeiss, LSM 510).

Embryos were harvested, fixed O/N at 4 °C in 4% PFA in PBS, rinsed in PBS, cryoprotected in 30% sucrose O/N at 4 °C, then embedded in OCT compound and cryosectioned at 12 μm per section. Slides were blocked for 1hr with 10% fetal bovine serum (FBS)/PBS and incubated O/N in 0.1% bovine serum albumin (BSA) with primary antibodies (Ab). Primary Abs were: PBX1 (Cell Signalling, #4342, 1:200); FLAG M2 (Sigma, F1804, 1:500). Primary Ab binding was detected by AlexaFluor-conjugated secondary Abs (Invitrogen) at 1:1000 dilution. Specifically, a donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary Ab (AlexaFluor™ 647; #A-31573) was used to detect PBX1, and a donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary Ab (Alexa Fluor™ 488; #A-21202) was used to detect FLAG M2. Nuclei were stained with DAPI (Sigma). Fluorescence imaging was performed using a Leica SP5 confocal microscope.