Prolong gold antifade mountant reagent

ASM cells were plated on cover slips in 12-well plate. After grown to confluence, cells were treated with 300 μM Sul-121, with or without 15% CSE or 10 μg/ml LPS (Sigma, L-2880), for 2 hours. Cells were then fixed with a solution containing 4% paraformaldehyde and 4% sucrose for 15 min at room temperature, followed by treatment with 0.3% Triton X-100 for 5 min at room temperature. Cells were blocked for 1 hour at room temperature with PBS containing 5% bovine serum albumin and 2% donkey serum. Cells were then incubated overnight at 4 °C with primary antibodies against Nrf2 (Abcam, ab31163, 1:100) and p65 (Cell Signaling, #3033S, 1:20). The next day, cells were washed with PBS and incubated with secondary antibodies (1:500) for 1 hour at room temperature. After wash with PBS, nuclei were stained with Hoechst (Invitrogen, H3570, 1:10000) for 5–10 sec, immediately followed by two quick and four 10 min washing steps with ultra-pure water. After staining, coverslips were mounted using ProLong® Gold Antifade Mountant reagent (Life Technologies, P36930) and imaged using an Olympus AX70 microscope equipped with digital image capture system (ColorView Soft System with Olympus U CMAD2 lens, Olympus Corporation, Tokyo, Japan). The background corrected fluorescence measurements were performed with Image J 1.48v58 (link). Data represent from 4–5 experiments.

BV2 cells (5 × 10⁵ cells) were seeded on sterile coverslips and pretreated with 20 μM ISO for 1 h and added with 20 μM Aβ_25–35 for 24 h. After washing with PBS three times, the cells were fixed in 4% paraformaldehyde for 10 min at room temperature. After washing with PBS three times, cells on coverslips were permeabilized in 0.2% (v/v) Triton X-100 for 5 min at room temperature. Cells were washed again with PBS, the coverslips were blocked with PBS containing 5% BSA for 5 min and incubated with an antibody against p-NF-κB (sc-166748, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted with PBS containing 5% BSA (1:500) for 1 h at room temperature. After washing with PBS three times, the coverslips were incubated with a secondary antibody Alexa Flour 488 (1:500, Invitrogen, Carlsbad, CA, USA) for 30 min and counterstained for nuclei with Hoechst 33,342 for 10 min. After washing with PBS three times, the coverslips were mounted using Prolong Gold antifade Mountant reagent (Life Technologies, Carlsbad, CA, USA). Each coverslip was analyzed on the Nikon Eclipse Ti fluorescence microscope (Nikon Instruments Inc., New York, NY, USA).

A tissue cut of up to 10 mg from tumour samples was subjected to gentle pipetation with 1 ml pipete, in order to separate the cells. Tumour and CSF samples were centrifuged at 600 × g for 7 min. The supernatant fluid was gently removed, and 500 μl of PBS solution were added to the sediment cells. These steps were then repeated. After the second centrifuge, the supernatant fluid was gently removed, and 10 μl of a solution containing methanol and acetic acid (3:2) were added to the cells, followed by rapid transfer of 20 μl of the cells to the center of a slide. After 7 min, the slide was transferred through an ethanol gradient (70%, 85%, 100% for 2 min each). Two drops of NucBlue® Fixed Cell Stain ReadyProbes® Reagent (Thermo Fisher Scientific, Waltham, MA, catalog no. R37606) were diluted in 1 ml phosphate-buffered saline, and 200 μl of this 4′,6-diamidino-2-phenylindole (DAPI) solution were added to the slide. After 5 min, unincorporated DAPI solution was removed, and 10 μl of ProLong® Gold Antifade Mountant Reagent (Thermo Fisher Scientific, catalog no. P36930) were added.