Pa5 27436

The hippocampus and prefrontal cortex (PFC) were dissected from male pups in all groups (P56). Tissue was homogenized in ice-cold radio-immune precipitation assay buffer with protease inhibitor, followed by protein isolation and quantification in a detergent-compatible assay. Equal concentrations of protein (30 µg) were loaded onto 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA for 1 h and then incubated overnight at 4 °C with the following primary antibodies: anti-GFAP (1:1000; MAB360; Sigma-Aldrich, Burlington, MA, USA), anti-Iba1 (1:500; PA5-27436; Thermo Fisher, Waltham, MA, USA), or anti-β-actin (1:5000). Then, the membranes were washed with 1× Tris buffered saline with Tween-20 (TBST) buffer (5 min, three times) and incubated with horseradish peroxidase-bound secondary antibody for 1 h. After washing with TBST, Western blot images were acquired using a Chemi Doc system (Bio-Rad, Hercules, CA, USA). Images were analyzed using ImageJ software (version 1.5; National Institutes of Health, Bethesda, MD, USA).

Cells were fixed in methyl alcohol and permeabilized with 0.3% Triton X-100 in phosphate-buffered saline (PBS). After blocking in 10% bovine serum albumin (BSA), cells were incubated overnight at 4 °C with the following primary antibodies: anti-GFAP (MAB360; EMD Millipore, Burlington, MA, USA), anti-aldh1L1 (NBP2-50045; Novus Biologicals, Littleton, CO, USA), anti-nestin (ab92391; Abcam), anti-Oct4 (ab18976; Abcam), anti-neuronal nuclear protein (NeuN) (MAB377; EMD Millipore), anti-MAP2 (M9942; Sigma-Aldrich, St. Louis, MO, USA and ab32454; Abcam), anti-ionized calcium-binding adaptor molecule 1 (Iba1) (PA5-27436; Invitrogen, Carlsbad, CA, USA and NB100-1028; Novus Biologicals), anti-bromodeoxyuridine (BrdU) (B5002; Abcam), anti-Ki67 (ab15580; Abcam), anti-BDNF (ab108319; Abcam), and anti-nerve growth factor (NGF) (ab52918; Abcam). After treatment with primary antibodies, the cells were washed with PBS and then incubated with secondary antibodies conjugated with Alexa488, Alexa555 (A11001, A22428; Invitrogen), and Alexa647 (ab150135; Abcam) for 1.5 h at room temperature. After washing with PBS, immunostained cells were mounted with VECTASHIELD^® Antifade mounting medium with DAPI (H-1200; Vector Laboratories, Burlingame, CA, USA) and observed with a confocal microscope (FV31-S; Olympus, Tokyo, Japan).

Table 2 shows the series of antibodies used in this study. The antibody against type IV collagen was used to demarcate the basal lamina (BL) of neurons, blood vessels, and epithelium. We used a polyclonal antibody (1:25, goat ab, AB769, Millipore). For residents’ macrophages, we used antibody against IBA1 (polyclonal, 1:100, rabbit, PA527436 from Invitrogen). Specificity was proven by IBA1 antibody blotting (36 (link)). Fractalkine antibody was a monoclonal antibody (1:100, mouse, MAB3651, R&D Systems). This antibody specificity antibody was verified in western blotting experiment (37 (link)). Information about the other primary and secondary antibodies is shown in Table 2.

After clearing, the brain was rinsed with PBST (1 × PBS with 1% Triton X-100, 0.02% sodium azide) at RT for 3-4 times. The brain was then embedded in 4% agarose (Sigma-Aldrich), cut including the neural probe into blocks of 1 mm (length) ×2 mm (width) ×1 mm (height). The brain tissue was incubated with mouse anti-FOX3 (1:500, SIG-39860, BioLegend), rat anti-GFAP (1:300, 13-0300, Invitrogen), and rabbit anti-Iba1 (1:300, PA5-27436, Invitrogen) for 4 days at RT. After incubation, brain tissue was rinsed in fresh PBST to 3 times before its overnight incubation at RT. The brain tissue was subsequently incubated with donkey anti-mouse Alexa Fluor 488 (1:300, Invitrogen), donkey anti-rat Alexa Fluor 594 (1:300, Invitrogen), goat anti-rabbit Alexa Fluor 647 (1:300, Invitrogen) for 3 days at RT. Then this brain tissue was rinsed with PBST up to 3 times at RT. Finally, this brain tissue was incubated in a refractive index matching solution for 30 min, and then images were scanned using a fluorescent microscope. The minimum animal numbers of each experiment (n = 6) were determined based on previous publications^{14 (link),53 (link)}.

At 24 h after ARDS induction, three rats from each group were deeply anesthetized with pentobarbital sodium (40 mg/kg), followed by transcardial perfusion with 100 mL PBS and 300 mL 4% paraformaldehyde (PFA; C104190; Aladdin, Shanghai, China). The hippocampal and cortex tissues were fixed in 4% PFA for 12 h and embedded in optimal cutting temperature compound (Sakura, CA, USA). Hippocampal tissue slices (50 μm) were renatured in citrate buffer (pH 6.0) at 108°C for 5 min. For immunohistochemistry (IHC), the renatured slices were pretreated with 1% H₂O₂ at room temperature (RT) for 15 min. After three PBS washes, the slices were masked with 10% NGS for 1 h in PBS. Slices were incubated with anti‐NLRP3 (SAB5700723; Sigma‐Aldrich) at 4°C for 24 h, followed by incubation with biotin‐labeled goat antibody against rabbit immunoglobulin G (IgG; 1:250; A0277; Beyotime) for 2 h and avidin–biotin complex for 1 h at RT. After three PBS washes, the slices were immersed in 3,3′‐diaminobenzidine solution at 37°C for 10 min.
For immunofluorescence, after masking with NGS, the slices were probed with an antibody against Iba‐1 (1:1000; PA5‐27436; Invitrogen, CA, USA) at 4°C for 24 h, followed by incubation with Alexa 488‐conjugated secondary antibody against rabbit IgG (1:250; A0428; Beyotime). Tissue slices were stained with DAPI before sealing.