Tcs sp5 fluorescence microscope

Frozen muscular sections were transferred to glass slides and fixed in 4% paraformaldehyde for 15 minutes at 4 °C. Thereafter, samples were blocked with PBS containing 1% BSA for 15 minutes at room temperature. After blocking, sections were incubated for 120 minutes at room temperature with NOS1 primary antibody (described in western blot analysis) in the same buffer solution, and then with Alexa Fluor^® 488 conjugate secondary antibody (Invitrogen, Oregon, USA) for 60 minutes at room temperature. Finally, sections were rinsed in PBS, mounted in Vectashield conjugated 4,6-diamidino-2-phenylindole (DAPI) in order to identify the nucleus (Vector Laboratories, CA, UK), and observed with a confocal Leica TCS SP5 fluorescence microscope.

To study the subcellular localization of OsSYF2, GFP was fused to the C terminus of OsSYF2 in the pBI221 vector. The plasmids were co-transformed into rice protoplasts, which were released from 10-day-old rice seedlings, with the nuclear marker construct AtH2B-mCherry via the polyethylene glycol (PEG 4000) method (Bart et al. 2006 (link)). After culturing overnight, the transformed protoplasts were observed with a Leica TCS SP5 fluorescence microscope.

The cells were incubated with DMSO (Wako Pure Chemical) or a DMSO solution of sample A (extract of twig of Caucasian beech, 100 μg/mL) (1/100 volume) for 4 h. Then, the cells were washed and stained with Hoechst 33342 (Thermo Fisher Scientific), Annexin V-FITC (PromoKine, Heidelberg, Germany), and EtD-III (PromoKine), as previously described [23 (link),24 (link)]. For immunostaining, the NFκB p65 antibody (Santa Cruz, Dallas, TX, USA) and anti-Nrf2 antibody (Abcam, Cambridge, UK) were used, and the staining was basically processed according to previously reported method [25 (link)]. The Leica (Wetzlar, Germany) TCS SP5 fluorescence microscope was used. For immunoblot analysis, THP-1 cells were incubated with or without sample A for 4 h, as described above. The cells were then lyzed and analyzed by immunoblotting, as previously described [26 (link),27 (link),28 (link)]. As an antibody, anti-Caspase-3 (Cell signaling technology, Danvers, MA, USA), IκB-α antibody (Cell signaling technology), or anti-β-actin (Sigma-Aldrich) was used.