Diaphot fluorescence microscope

Coronal sections of temporal cortex (n= 3–5) sections from different brain were washed with 0.1% PBS-TritonX-100for 20 min. For most antibodies used in this study, antigen retrieval was done using sodium citrate buffer with 0.1% Triton-X. Sections were blocked with blocking solution containing 10% donkey serum for 1 h at room temperature. Sections were incubated at 4 °C with the respective primary antibodies as shown in the figure legends. The following primary antibodies were used :mouse anti-amyloid beta (6E10) (1:250), mouse anti-phospho-tau (1:250), mouse anti-GMF (1:200), mouseanti-NLRP3 (1:250), rabbit anti-capase-1(1:200), rabbit anti-IL-1β (1:200), mouse anti-IL-18 (1:200), rabbit anti-LC3 (1:200), rabbit anti-SQSTM1/P62 (1:250) and rabbit anti-LAMP1(1:250). The sections were washed three times followed by incubation with the respective Alexa Fluor-488 (green) and/or Alexa Fluor-568 (red) tagged secondary antibodies (1:500) for 1h at room temperature. Thereafter sections were washed and mounted with a cover slip using Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (H-1200; Vector Laboratories, Inc., CA, USA). Images were acquired on a fluorescence Nikon (DIAPHOT) fluorescence microscope. Staining intensity and area measurement was undertaken using the MetaMorph image analysis software.

hBE cells growing on 2-well chamber slides were incubated with fluorescent DNA dyes YoYo-1 (1.0 μmol), SYTOX Green (10 μmol), or DAPI (0.1 μg/mL) in HBSS solution containing 10 mmol HEPES buffer for 15 minutes before exposure to 100 μg/mL Alternaria in HBSS solution for 30 minutes. Fluorescent dye bound to eDNA was visualized with a Nikon Diaphot fluorescence microscope at excitation/emission wavelengths of 358 nm/460 nm (DAPI), 491 nm/509 nm (YoYo-1), and 504 nm/523 nm (SYTOX Green) with a 40× fluorescence objective (Nikon, Tokyo, Japan).