Prolong antifade reagent

Briefly, the slides containing SH-SY5Y cells were washed twice for 10 min each in 0.01 M PBS and incubated for 1 h in blocking solution containing 2% normal bovine serum (Santa Cruz Biotechnology), according to the antibody treatment, and 0.3% Triton X-100 in PBS. After blocking, the slides were incubated overnight at 4 °C with anti-p-JNK, anti-BACE-1, and anti-Nrf2, (Santa Cruz Biotechnology) and mouse monoclonal anti-8-Oxo-G (Millipore) antibodies diluted 1:100 in blocking solution. Following this, the slides were incubated for 2 h with the fluorescein isothiocyanate FITC/TRITC-labeled secondary antibodies (1:50) (Santa Cruz Biotechnology). The slides were then counterstained with 40,6-diamidino-2-phenylindole (DAPI) for 10 min and mounted with the Prolong Anti-fade Reagent (Molecular Probe, Eugene, OR, USA). Staining images of the double immunofluorescence were examined using a confocal laser-scanning microscope (Flouview FV 1000, Olympus, Japan).

Paraffin embedded tissue samples were sectioned and stained with hematoxylin and eosin for histomorphological analysis. Different antigen unmasking methods where used on tissue slides for immunohistochemical staining, which was performed using anti-CD163 (Leica/Novocastra), anti-Siglec-1 (Novus Biologicals) and anti-pSTAT1 (Cell Signaling Technology). Sections were then incubated with biotin-conjugated polyclonal anti-mouse or anti-rabbit immunoglobulin antibodies, followed by the streptavidin-biotin-peroxidase complex (ABC) method (Vector Laboratories). Finally, sections were counter-stained with hematoxylin. Slides were scanned with the Panoramic 250 Flash II (3DHISTECH). Virtual slides were automatically quantified for macrophage distribution as previously described (Souriant et al., 2019 (link)). Immunofluorescence staining of the sections was performed as described above, and followed by anti-mouse IgG isotype specific or anti-rabbit IgG antibodies labelled with Alexa488 and Alexa555 (Molecular Probes). Samples were mounted with Prolong Antifade reagent (Molecular Probes) and examined using a 60x/1.40N.A. objective of an Olympus spinning disk microscope.

The morphological evaluations were performed as previously described with some modifications [13 (link),21 (link)]. The prepared tissue slides were washed twice for 10 min in 0.01 M PBS, followed by incubation for 1 h in a blocking solution containing 2% normal serum according to the antibody treatment and 0.3% Triton X-100 in PBS. After blocking, the slides were incubated overnight at 4 °C in the primary antibodies (mouse polyclonal phosphorylated c-JUN N-terminal Kinase (p-JNK), rabbit polyclonal anti-p-NFκB, mouse polyclonal TNF-α, rabbit polyclonal anti- ionized calcium-binding adaptor molecule 1 (Iba-1), mouse monoclonal post synaptic density-95 (PSD-95) from Santa Cruz Biotechnology and mouse monoclonal 8-Oxoguanine (8-OxoG) from Millipore) and diluted 1:100 in blocking solution. After incubation with primary antibodies, the sections were incubated for 2 h in the secondary tetramethylrhodamine (TRITC)/fluorescein isothiocyanate (FITC)-labeled antibodies (1:50) (Santa Cruz Biotechnology, Dallas, Texas, USA). After secondary antibody incubation, tissue slides were washed twice for 5 min. Slides were mounted with 4′, 6′-diamidino-2-phenylindole (DAPI) and Prolong Antifade Reagent (Molecular Probe, Eugene, OR, USA). Then, slides were examined using a confocal laser-scanning microscope (Flouview FV 1000, Olympus, Tokyo, Japan).

Cells, plated on High Precision 1.5H 12-mm coverslips (Marienfeld), were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min or pre-extracted (when using pRVSF-KNL1 or mCherry antibodies) with 0.1% Triton X-100 in PEM (100 mM Pipes, pH 6.8, 1 mM MgCl2 and 5 mM EGTA) for 1 minute before addition of 4% PFA for 10 minutes. Coverslips were washed with PBS and blocked with 3% BSA in PBS + 0.5% Triton X-100 for 30 min, incubated with primary antibodies overnight at 4°C, washed with PBS and incubated with secondary antibodies plus DAPI (4,6- diamidino2-phenylindole, Thermo Fisher) for an additional 2-4 hours at room temperature in the dark. Coverslips were then washed with PBS and mounted on glass slides using ProLong antifade reagent (Molecular Probes). All images were acquired on a DeltaVision Core or Elite system equipped with a heated 37°C chamber, with a 100x/1.40 NA U Plan S Apochromat objective using softWoRx software (Applied precision). Images were acquired at 1x1 binning using a CoolSNAP HQ or HQ2 camera (Photometrics) and processed using softWoRx software and ImageJ (National Institutes of Health). All immunofluorescence images displayed are maximum intensity projections of deconvolved stacks and were chosen to most closely represent the mean quantified data.