The procedures used in this study were approved by the Ethics Committee on the Use of Animals of the Oswaldo Cruz Foundation (CEUA licenses n°002-08, 36/10 and 054/2015). Male Swiss Webster mice weighing between 20-30 g, provided by the Institute of Science and Technology in Biomodels (ICTB) of the Oswaldo Cruz Foundation (FIOCRUZ), were used for the experiments. The animals were kept in ventilated isolators in the Pavilhão Ozório de Almeida's vivarium, and water and food were available ad libitum. They were exposed to a 12 h/12 h light and dark cycle. 

1. Preparation of sodium oleate solution

1. Use oleic acid to prepare a 100 mmol/L of sodium oleate stock solution in any sterile tube or glass flask.
   NOTE: A 50 mL (final volume) solution was prepared for the present work, but the volume must be adjusted as per the experimental need. The solution must be always prepared in sterile tubes or glass containers.
   1. First, add NaOH tablets or solution in ultrapure water to elevate the pH. A pH value of 12-13 is recommended for a 25 mL volume.
      NOTE: Alternatively, Tris base can be used to prepare the Tris-oleate solution.
   2. Add the oleic acid (see Table of Materials) very slowly, drop by drop, under constant agitation in an ultrasonic bath at 37 °C.
      NOTE: If oleic acid precipitation occurs, start all over from the beginning.
   3. Once oleic acid is completely dissolved, carefully adjust the pH to 7.4, drop by drop under stirring, with ultra-pure diluted HCl and then adjust to the final volume of 50 mL.
      ​NOTE: Freshly prepare the working oleate solutions. Alternatively, the solution may be aliquoted, stocked, and maintained at -20 °C in a nitrogen-enriched environment to avoid oxidation for no longer than a month. Avoid frozen-refrozen cycles.

2. Induction of lung injury by oleic acid

1. Perform the intratracheal administration of oleic acid.
   1. Anesthetize the mice using 5% isoflurane with 2 L/min of O₂ employing a veterinary anesthetic vaporizer (Figure 1A). Remove the fur at the incision area with depilatory cream and disinfect the area with three alternating rounds of betadine scrub and alcohol using sterile gauze. Confirm the depth of anesthesia by toe pinch.
      NOTE: Use sterile gloves and instruments during the procedure. Use a drape to cover the animal and expose only the incision site. Perform the experiment in a biological safety cabinet to avoid isofluorane escape to the environment. Analgesics are not administered as they may inhibit the inflammatory response.
   2. After anesthesia, lay the animal in a dorsal decubitus position and make an incision (0.5-1 cm) in V-shape at the thyroid level. Gently displace the thyroid to expose the trachea (Figure 1B) and inject 50 µL of the prepared oleate solution (step 1).
      NOTE: The mice were divided into two groups, with eight animals in each group. The lung injury group receives sodium-oleate solution at 25 mM (1.25 µmol), and the control group receives 50 µL of sterile saline by instillation into the trachea of each mouse with an insulin syringe (volume 300 µL, 30 G) (Figure 1C).
   3. Suture the mice's incision site with a synthetic non-absorbable monofilament suture, return it to their cage, and monitor it until complete recovery from the surgery. During all procedures, maintain the animals on a heating pad at 37 °C.
      NOTE: Mice usually take up to 15 min to recover from surgery.
2. Perform intravenous administration of oleic acid.
   1. After anesthesia (step 2.1.1, Figure 2A), inject intravenously into the orbital plexus by inserting the ultrafine needle (see Table of Materials) in the medial canthus of the eye socket (Figure 2B).
      ​NOTE: The mice were divided into two groups, with eight animals in each group. Each group receives 100 µL of the sodium-oleate solution at 10 µmol of OA per animal, while the control group receives 100 µL of sterile saline.
3. After the surgery, monitor the animals daily for adverse reactions. Humane endpoints for euthanasia include adverse reactions, convulsions, and coma.

3. Bronchoalveolar lavage fluid collection (BALF)

1. Euthanize the mice with an intraperitoneal lethal dose of ketamine (300 mg/Kg) and xylazine (30 mg/Kg) (see Table of Materials).
2. Lay the animal in the dorsal decubitus position, make an incision of approximately 1 cm with surgical scissors in the animals' anterior region, expose the trachea and make a small cut to introduce an intravenous catheter (20 G).
3. Connect the catheter to a 1 mL sterile syringe, slowly and gradually inject 0.5 mL of sterile saline into the lungs, and then aspirate the fluid from the BALF with the same syringe. Repeat it 3-5 times, and transfer it to a sterile microtube, placing them in ice.
   ​NOTE: The samples can be stored at -20 °C for up to 6 months.

4. Total and differential cell analysis in BALF

1. For total cell count, dilute 20 µL of BALF in 180 µL (10x dilution) of Turk's solution (see Table of Materials). Perform the counting using a Neubauer chamber under an optical microscope with a 40x objective.
2. For differential count, put 100 µL of BALF in the cellular funnel containing slides and centrifuge it at 22.86 x g for 5 min at 4 °C in a cytocentrifuge, and stain with May-Grunwald (15%, pH 7.2)-Giemsa (1:10) (see Table of Materials). Proceed with cell count in a light microscope with immersion objective.

5. Determination of total protein in BALF

1. Determine the total BALF supernatant protein by a commercial protein quantification kit and read the absorbance at 562 nm using a spectrophotometer following the manufacturer's instructions (see Table of Materials).

6. Enzyme immunosorbent assays

1. Centrifuge BALF at 1,200 x g for 10 min at 4 °C. Then collect the supernatant with a pipette and store it at -80 °C for assays of TNF-α, IL-1β, IL-6, and PGE2^15,23,25.
   NOTE: The centrifugation in step 6.1 makes the BALF cell-free.
   1. Perform the cytokines assays on cell-free BALF using a commercial ELISA kit according to the manufacturer's instructions. Carry out the PGE2 assay using an enzyme immunoassay (EIA) kit following the manufacturer's instructions (see Table of Materials).

7. Lipid body staining and counting

1. Fix the leukocytes on cytospin slides using 3.7% formaldehyde in Ca²⁺, Mg²⁺ free Hank's buffered salt solution (HBSS, pH 7.4) and stain with 1.5% OsO₄ while still moist³ (see Table of Materials). Then, count the lipid bodies per cell in 50 consecutive leukocytes from each slide using the oil-immersion objective lens of the microscope.

8. Statistical analysis

1. Perform statistical analysis using graphing and statistics software (see Table of Materials). Express the results as mean ± SEM and analyze by one-Way Anova followed by a post-test Newman-Keuls-Student²⁶. Consider the differences significant when P < 0.05.