Alexa fluor 594 or 488 conjugated secondary antibodies

Oocytes were fixed in 4% paraformaldehyde in PBS for 30 min and permeabilized in PBS containing 0.3% Triton X-100 for 20 min. After being blocked with 1% bovine serum albumin in PBS, the oocytes were incubated with primary antibodies for 1 h and sequentially labeled with Alexa Fluor 594- or 488-conjugated secondary antibodies (Molecular Probes) and 4′,6-diamidino-2-phenylindole (DAPI) for 30 min. Imaging of oocytes after immunofluorescence was performed on a LSM710 confocal microscope (Zeiss). The antibodies used are listed in Supplementary Table S1.

OCIC spheroids were harvested and placed on glass slides, fixed in paraformaldehyde (4°C, 30 min), permeabilized with PBS that contained 0.3% Triton X-100 (PBST), and incubated with blocking buffer (PBST containing 5% bovine serum albumin). Spheroids were sequentially probed with the primary antibodies and Alexa Fluor 594- or 488-conjugated secondary antibodies (Molecular Probes). Slides were mounted using VectaShield with 4′, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories). Digital images were acquired using an epifluorescence microscope (Nikon Eclipse 80i) with 4-100X objectives.

Cells grown on glass coverslips were fixed in 4% formaldehyde supplemented with 0.1% Triton X-100 for 15 min at room temperature prior to permeabilisation in 0.5% Triton X-100 for 5 min. After blocking with 3% BSA in PBS containing 0.05% Tween 20, the cells were stained for 1 h in blocking solution with antibodies. Primary antibody detection was achieved via incubation with anti-rabbit or anti-mouse Alexa Fluor 594- or 488-conjugated secondary antibodies (Invitrogen) for 45 min at room temperature. The slides were mounted in DAKO mounting medium supplemented with DAPI (Sigma) and examined at a magnification of 63× via fluorescence microscopy (Zeiss Axio Observer Z1). The images were captured with an ORCA-ER camera (Hamamatsu). The microscope and camera parameters were adjusted for each series of experiments to avoid signal saturation. Image processing and analysis were performed using ImageJ software.

Cells were washed in PBS before fixation with 4% paraformaldehyde for 15 min and then permeabilized with 0.2% Triton-X 100 for 10 min at room temperature (RT). After blocking with 3% BSA for 1 hr, cells were incubated with primary antibody at the recommended dilution, followed by Alexa-Fluor 594- or 488-conjugated secondary antibodies (Invitrogen, Thermo Fisher Scientific) at a 1:100 dilution for 1 hr. Nuclei were labeled with Hoechst-33342 (Thermo Fisher Scientific) before cells were mounted onto a microscope slide using Mowiol® 4-88. Finally, IF-staining images were captured with a Zeiss Axio Observer microscope. The primary antibodies used in this study are listed in Table S2.

The procedures used were the same as those we reported previously (Pan et al., 2022 (link)). Briefly, mouse brain tissues were removed, infiltrated with paraffin, and cut into serial 4 μm sections. The slices were processed using an IHC Detection System Kit (ZSGB‐BIO, PV‐6001/PV‐6002). The signal was developed using DAB. For immunofluorescence staining, the samples were stained with corresponding Alexa Fluor 594‐ or 488‐conjugated secondary antibodies (1:1000; Invitrogen). Nuclei were stained with DAPI (1 μg/mL, BioFroxx, 1155MG010) for 5 min. The sections were incubated with primary antibodies against Cox IV (1:500, Abcam, ab16056), TH (1:1000, Abcam, ab117112), or K182Hcy (1:500, Abmart) at 4°C overnight. Images were captured using an Olympus DP80 microscope equipped with TH4‐200 and U‐HGLGPS light sources. The levels of immunoreactivity were determined by optical density analysis using ImageJ plus the IHC Profiler plugin and measured using the procedure in the literature as follow: optical density score = percentage contribution of high positive × 4 + percentage contribution of positive × 3 + percentage contribution of low positive × 2 + percentage contribution of negative × 1 (Seyed Jafari & Hunger, 2017 (link)).

The frozen sections from human nasal tissue were blocked with 10% goat serum for 30 min and stained first with IL‐19 (1:50; Abcam) and MMP‐9 antibodies (1:50; Abcam) overnight at 4°C, separately. HNECs were fixed with 4% paraformaldehyde, blocked in 1% bovine serum albumin for 30 min, and then stained first with MMP‐9 or p65 antibodies (1:500; Cell Signaling Technology) overnight at 4°C. Subsequently, the frozen tissue sections and HNECs were incubated with Alexa Fluor 594‐/or 488‐conjugated secondary antibodies (1:1000; Invitrogen) for 1 h at room temperature. Finally, the slices were stained with 4′,6‐diamidino‐2‐phenylindole (1:30,000; Biolegend) to mark the nuclei, and sealed with antifade mountant (Invitrogen). IF was examined at ×400 magnification under a microscope (Leica), or at ×630 magnification in a confocal microscope (Carl Zeiss).