Ammonium hydroxide solution

Tetraethyl orthosilicate (≥99.0%, 86578), ammonium hydroxide solution (29%, 221228), (3-aminopropyl)triethoxysilane (≥99.0%, 440140), (3-mercaptopropyl)triethoxysilane (≥80%, 63797), (3-cyanopropyl)triethoxysilane (≥98%, 374156), and the buffer solution used for the zeta potential measurement (potassium dihydrogen phosphate/di-sodium hydrogen phosphate, 12161706) were purchased from Sigma Aldrich. Ethanol (EtOH, 99.9%, PC80101.9200) was supplied by Atlantic Labo. Carbon/copper grids were obtained from Delta Microscopies. All chemicals were used without further purification. Aqueous solutions were prepared using deionized water, obtained from a Type 1 Milli-Q water purification system (18.2 MΩ cm).

Staining was performed as previously described^{40 (link),65 (link)}. Lung samples were fixed in a 10% formalin solution (Cat: HT501320-9.5L, Sigma–Aldrich) and agitated overnight. The samples were subjected to sequential dehydration in 70%, 80%, 90%, and 100% ethanol over the following 4 days. The lung samples were then immersed in xylene for 1 h; this process was performed three times. The xylene-soaked tissue was then placed in a paraffin-containing glass vessel and placed in a 60 °C oven overnight to allow paraffin infiltration into the tissue. The next day, paraffin blocks were constructed using an embedding machine (Shandon Histocentre 3, Thermo Fischer Scientific). Tissue sections were prepared using a Semi-Automated Rotary Microtome (Cat: RM2245, Leica Biosystems, Wetzlar, Germany). Sectioned tissues were mounted on non-adherent slides and air-dried on a 50 °C heat plate overnight. Paraffin on the slides was removed via triple immersion in xylene at 7-min intervals. Dehydrated lung tissue sections were rehydrated via sequential immersion in bottles containing 100%, 90%, 80%, and 70% ethanol at 3-min intervals. The sections were then incubated in hematoxylin (Cat: GT0111, Glentham, Life Sciences Ltd., Wiltshire, UK) for 1 min for nuclear staining. The slides were then rinsed with tertiary distilled water and incubated in a 0.1% ammonium hydroxide solution (Cat: 221228, Sigma–Aldrich) for 1 min to complete nuclear staining. After the bluing reaction, the slides were washed with tertiary distilled water and incubated in eosin (Cat: EM000G-5, Cancer Diagnostics, NC, USA) for 1 min to stain the cytoplasm. After cell staining, Canadian balsam solution (Cat: C1795, Sigma–Aldrich) was applied directly onto the lung tissue, and a cover glass was placed over it. The 100× images of the lung sections were obtained using an Olympus BX41TF light microscope (Olympus Corporation, Tokyo, Japan). The immune cell-infiltrated area was quantified using ImageJ software (National Institutes of Health).