Mca74g

Lung and colon tissues embedded in paraffin were sectioned and rehydrated. The tissues were antigen retrieved in tri-sodium citrate buffer containing 0.2% tween 20 (pH 6.0) with an autoclave for 1 min. 3% H₂O₂ solution was treated for 10 min to block endogenous peroxidase activity. The tissues were washed in PBS and blocked with 1.5% bovine serum albumin (BSA) containing 0.2% triton X-100 for 1h at room temperature. Rat anti-mouse CD11b primary antibodies (MCA74G) (Abd Serotec; 1:200) and biotinylated anti-Rat antibodies (BA-9401) (Vector laboratories; 1:1,000) were diluted in 0.5% BSA. The slides were overnight incubated with primary antibodies at 4°C and incubated with secondary antibodies for 1h at room temperature. The avidin-biotin complex kit (Vectastain ABC kit; PK-6102) (Vector Laboratories, Burlingame, CA, USA) and diamminobenzidine-HCl (DAB) kit (SK-4100) (Vector Laboratories, Burlingame, CA, USA) were used to visualize the tissue. The slides were mounted and observed under the microscope.

CEABAC lung cells from untreated or bleomycin‐treated mice were isolated by elastase digestion and sorted as previously described for rat alveolar epithelial cells (Gonzalez et al. 2009). Briefly, cells were initially treated with human IgG (50 μg/mL; Sigma) to block non‐specific binding of mouse monoclonal primary antibody. CEACAM6 (clone 9A6) antibody was directly labeled with Alexa 488 using Zenon technology (Invitrogen, as recommended). Cells were stained with labeled CEACAM6 antibody (60 μL) and rat anti mouse CD11b (1:50) to label cells of myeloid origin (AbD Serotec, MCA74G, Raleigh, NC). Cells were sorted and collected on an Aria II FACS instrument (BD Biosciences, San Jose, CA). In some cases collected cells were sorted directly into RLT Bufffer (Qiagen Inc, Valencia, CA) for future use, while in other cases cells were fixed in 50% ethanol for cell cycle analysis and stained with a solution containing propidium iodide (PI, 20 mg/mL), DNase‐free RNase A (0.2 mg/mL), and 0.1% Triton‐X100 in calcium, magnesium‐free PBS. All analyses were done using Flow Jo software (Tree Star Inc., Ashland, OR).

Brain sections were washed (4 × 3 min in 0.15 M phosphate buffer (PB)), blocked in a PB solution of 6% normal goat serum (NGS) and 0.4% Triton-X for 1 h, then placed in a primary antibody PB solution containing 0.4% Triton-X and 3% NGS for 48 h at 4 °C. Primary antibodies consisted of Iba1 (Rabbit; Wako 019-19741; 1:2000) paired with CD68 (Rat; Bio-Rad MCA1957; 1:1000) or CD11b (Rat; Bio-Rad MCA74G; 1:1000). Following primary antibody incubation, sections were washed (4 × 3 min in PB), placed in a fluorescent secondary antibody PB solution of 0.4% Triton-X and 3% NGS for 2 h, removed from secondary, washed in PB (4 × 3 min), stained with Hoechst stain (Sigma-Aldrich, H6024 23491-45-4; 1:100) for 7 min, washed in PB (4 × 3 min), mounted on gelatin-coated slides, and coverslipped with Prolong Gold (Invitrogen, P36930). Secondary antibodies consisted of Alexa-Fluor Goat x Rabbit 647 (Invitrogen, A21245) and Goat x Rat 594 (Invitrogen, A11007) diluted at 1:2000. All steps were done at room temperature unless noted.