Af 400 na

The injury model used was that of a section of the spinal cord. To produce a mouse model of the 10^th thoracic vertebra (T10) complete transection SCI, C57BL/6 mice were anesthetized using 5% isoflurane in oxygen for induction and 2% isoflurane in oxygen for maintenance of anesthesia. The back fur was shaved and disinfected with Iodine Volts Swabs. An ~2 cm incision was made in the skin over T9–T11, and then the fat, fascia layer, and paravertebral muscles were separated successively to expose the T10 vertebra. T10 laminectomy was performed and T10 spinal tissue was completely transected using a sharp scalpel. In the Sham group, mice underwent the same surgical procedure except for the transection. Then, the muscle and skin were sutured layer by layer. Postoperative care was composed of disinfected wound treatment and manual bladder compression for voiding twice daily after SCI. Upon euthanasia was performed in a chamber filled with 99.9% CO₂ gas for 10 min, and damaged regions of spinal cord tissue were harvested immediately for follow-up experiments. In particular, mice were intraperitoneally injected with an anti-mouse IL1α neutralizing antibody (R&D systems, Minneapolis, MN, SA, AF-400-NA, 20 mg/kg) and IgG control (R&D systems, Minneapolis, MN, SA, AB-108-C, 20 mg/kg) once per day for one week from the 1^st day after surgery.

Neonatal lungs sections were collected and fixed with 4% PFA and stored. Samples were then heated to deparaffinize and rehydrated with xylene and ethanol.
For DAB immunohistochemistry slides were quenched with BLOXALL (Vector labs) and blocked. They were then incubated with IL-1α primary antibodies (R&D systems, AF-400-NA), followed by a biotinylated secondary antibody, VECTASTATIN. R.T.U. ABC Reagent, and finally stained with DAB peroxidase (Vector Labs). They were counterstained with methyl green. IL-1α staining was visualized using the Olympus IX83 microscope and Olympus DP80 camera at Å~40 magnification using Olympus CellSens software.
For immunofluorescence, antigen retrieval was performed with citric acid, tissues were permeabilized with 0.5% triton, quenched with glycine, and blocked. They were incubated with IL-1α (R&D systems, AF-400-NA) and pro-surfactant protein C (Millipore, AB3786) primary antibodies overnight followed by incubation with secondary antibodies (Alexa fluor anti-rabbit donkey 647 and anti-goat donkey 594) for 1 h. Staining was visualized using the Olympus IX83 microscope and Olympus DP80 camera using Olympus CellSens software.

Organoid culture protocol was adapted from Lukacs et al.^{39 (link)}. Briefly, sorted bPSC were counted and resuspended in a 1:2 mixture of Prostate Epithelial Growth Medium (PrEGM, Lonza) and Matrigel (growth factor reduced, Corning). The mixture was deposited in a ring around the edge of a refrigerated 12-well plate at 10⁴ cells/120 μL/well and allowed to solidify for 15 minutes at 37°C before addition of pre-warmed PrEGM with or without treatments. Growth medium with treatments was changed every 2–3 days before counting and harvesting on day 7. The following treatments were used with vehicle controls: Enzalutamide (Selleckchem), R1881 (Sigma-Aldrich), recombinant mouse IL-1RA (R&D Systems), recombinant mouse IL-1α (R&D), IL1-RA neutralizing antibody (#AF-480-NA, R&D) and IL-1α neutralizing antibody (#AF-400-NA, R&D). Intact organoids were released from Matrigel with Dispase (Gibco), fixed in 10% neutral buffered formalin followed by 70% ethanol, and embedded in HistoGel (ThermoFisher Scientific).