M3696

To explore the neurite outgrowth, the immunostaining was performed as follows. Cultured neurons were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at 4 °C and wash three times with PBS for 5 min each. To permeate the cell membrane, 1% Triton X-100 (Sigma-Aldrich) was used for 5 min at room temperature, followed by PBS washing. For blocking nonspecific binding, the sample was treated with 6% bovine serum albumin (BSA; Sigma-Aldrich) for 30 min. After washing with PBS, it was reacted with primary antibodies diluted in 1.5% BSA solution for 2 h at 37 °C. The following primary antibodies were used: anti-MAP2 (1:500, M3696, Sigma-Aldrich), anti-tau-1 (1:500, MAB3420, Merck Millipore, MA, USA), anti-beta-III-tubulin (1:500, T2200, Sigma-Aldrich). Secondary antibodies (Alexa Fluor 488 and 594, 1:500, A11001, A11008, A11012 and A11032, Invitrogen; Thermo Fisher Scientific) diluted in 1.5% BSA were loaded for 30 min at 37 °C. To characterize damage of a biomolecule on the NIR-illuminated surface, anti-laminin (1:500, L9393, Sigma-Aldrich) was used with the same procedure except that there is no cell fixation and permeabilization step. An inverted microscope (IX71; Olympus) with a digital camera (DP71; Olympus) was used to take fluorescence and phase-contrast images.

Motor neurons were cultured on glass coverslips for 12 days. Then, cells were rinsed twice in cold 0.01 M PBS (pH 7.4) and fixed in 4% (w/v) paraformaldehyde (Sigma-Aldrich, Milan, Italy) for 20 min at RT. After three washes in PBS, cells were blocked with 3% (w/v) BSA and 0.05% Triton-X (Bio-Rad, Milan, Italy) for 1 h at RT. Coverslips were then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal antibody against NCX1 (#π11-13, Swant, Bellinzona, Switzerland), rabbit polyclonal antibody against NCX3 (Dr. K. Philipson Laboratory, University of California, Los Angeles, CA, USA), mouse monoclonal antibody against SOD (#S2147, Sigma-Aldrich, Milan, Italy), mouse monoclonal antibody against p-Akt (#4051, Cell Signaling Technology Inc., Danvers, MA, USA), or rabbit polyclonal antibody against MAP2 (#M3696, Sigma-Aldrich, Milan, Italy). After three washes in PBS, coverslips were incubated in the dark with the corresponding secondary antibodies for 1 h at RT. Dapi was used to stain nuclei. Images were acquired by using a Zeiss LSM 700 laser (Carl Zeiss) scanning confocal microscope.