Fluorescence microscopy

SH-SY5Y cells cultured in DMEM/F12 medium were seeded for 24 h, then incubated with various concentrations of EP for 24 h followed by 6-OHDA for 6 h. Apoptotic, necrotic, and live cells were quantified using apoptosis/necrosis detection kits (blue, green, red, respectively; Abcam, Cambridge, UK) as described by the manufacturer. Fluorescence microscopy (Keyence, Tokyo, Japan) images were acquired and fluorescence intensity was quantified using ImageJ to determine apoptosis.

Primary hepatocytes were analyzed for MMP using the JC-1 staining method. In brief, primary hepatocytes were incubated with a JC-1 probe at a final concentration of 20 μM for 10 min at 37°C in the dark. Mitochondrial reactive oxygen species (mito-ROS) were measured using a final concentration of 5 μM MitoSOX™ red mitochondrial superoxide indicator (MitoSOX™, 5 μM, Invitrogen. M36008) at 37°C for 10 min in the dark. Cells were washed with PBS and immediately observed by fluorescence microscopy (Keyence, Osaka, Japan).

The mitochondrial membrane potential was assessed using the JC-1 MitoMP Detection Kit (DOJINDO #MT09) and Image-iT TMRM reagent (Thermo) as the manufacturer recommends. Early passage and replicative senescent ECs were incubated with TMRM reagent (1:1000) and MitoTracker Green (Thermo, 1:5000) for 30 min at 37 °C in the CO₂ incubator, followed by washing with PBS for 3 times. Subsequently cells were observed under fluorescent microscopy (Keyence). Some cells were treated with 150 μM spermidine for 48 h prior to the assay. Fluorescence intensities for TMRM and MitoTracker were measured using the image-J software, and the mitochondrial membrane potential was assessed by the TMRM fluorescence intensity normalized by MitoTracker intensity. For JC-1 assay, Early passage and replicative senescent ECs were incubated with 2 μM JC-1 for 1 h at 37 °C in the CO₂ incubator. After washing with HBSS twice, Imaging Buffer solution was added, followed by analysis of cells under fluorescence microscopy (Keyence). When the mitochondrial membrane potential is high, JC-1 forms red fluorescent “J-aggregates”, while JC-1 dye remain as monomer, exhibiting green fluorescence in mitochondria with low membrane potential. The mitochondrial membrane potential was assessed by the ratio of red/green fluorescence intensity. Results were confirmed by at least two independent experiments.

The cells were plated on chamber slides. The slides were fixed in 4% paraformaldehyde and washed extensively with PBS. The slides were stained using an In Situ Cell Death Detection Kit and TMR red (Roche Diagnostic, Indianapolis, IN, USA) according to manufacturer's instructions. The cells were mounted with an anti-fade mounting medium, and the TUNEL-positive cells were visualized by fluorescence microscopy (KEYENCE Corporation).

TG were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Tissue sections on slides were deparaffinized and incubated with anti-TRPV1 antibody (Alomone Labs, Jerusalem, Israel) and anti-CGRP antibody (Abcam) at 4 °C overnight. The slides were then incubated with Alexa Fluor 594 -conjugated anti-rabbit secondary antibody (Abcam) for TRPV1 and Alexa Fluor 488-conjugated anti-goat secondary antibody (Abcam) for CGRP at room temperature for 1 h. For histological analysis of alveolar bone tissues, decalcified and embedded sections were double-stained by anti-CGRP antibody and anti-PGP9.5 antibody (Novus Biologicals, Littleton, CO, USA) with appropriate Alexa Fluor-conjugated secondary antibodies. Slides were mounted with VECTASHIELD^® HardSet™ Mounting Medium with DAPI (Vector laboratories, Burlingame, CA, USA) and analyzed by fluorescence microscopy (Keyence Corporation).

Isolated hepatocytes or LSECs were cultured on a coverslip (AGC Techno Glass Co., Shizuoka, Japan), fixed with paraformaldehyde for 5 min and blocked with SuperBlock blocking buffer (Thermo Fisher Scientific). The cells were incubated with an E-cadherin antibody (BD Biosciences, San Jose, CA, USA) and VE-cadherin antibody (Abcam) at 1:200 for 1 h at room temperature. Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) were used as secondary antibodies for 1 h at room temperature with protection from the light. The cells were counterstained with DAPI and analyzed by fluorescence microscopy (Keyence Corporation, Osaka, Japan).
Mouse livers were mounted in Tissue-Tek O.C.T. compound (Sakura Finetek Japan Co Ltd., Tokyo, Japan) and frozen until preparation. Frozen sections were cut at 10-μm thickness and fixed in methanol for 10 min. Blocking was performed in PBS with 3 % bovine serum albumin (BSA). The slides were incubated with BMPER antibody (Abcam) at 1:200 in PBS overnight at 4 °C, and subsequently incubated with CD31 antibody (BD Pharmingen) at 1:100 in PBS for 1 h at room temperature. Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) were incubated at 1:200 in PBS for 1 h at room temperature. The slides were counterstained with DAPI and analyzed using fluorescence microscopy.