Rm 9106 r7

Analysis of S100 expression was performed using a rabbit polyclonal antibody Z0331 (1:400) for S100 (Dako; Glostrup, Denmark). Detection of MAPK activation was performed using a 1:100 dilution of an antibody against phospho-ERK (4370, Cell Signaling). Cell proliferation was detected using a 1:250 dilution of a rabbit monoclonal antibody against Ki67 (RM-9106-R7 Thermo Scientific, Waltham, MA, USA). IHC HA was performed using a 1:200) dilution of the HA.11 antibody. Apoptosis detection was performed using a 1:250 dilution of the cleaved caspase-3 antibody (9579 Cell Signaling). Controls were conducted without primary antibody on corresponding sections. Detection of HRP activity was performed using DAB (Cell Signaling). Sections were counterstained with hematoxylin.

For routine histology, primary tumors and lymph node metastases from BPT mice were fixed in paraformaldehyde and embedded in paraffin, and 5-µm sections were stained with hematoxylin and eosin. For lung histology, 4-µm sections of paraformaldehyde inflation– fixed and paraffin-embedded lungs were stained with hematoxylin and eosin. Immunohistochemical analyses were performed as described [16 (link),17 (link)]. The sections were incubated with antibodies recognizing Ki67 (RTU (ready-to-use), RM-9106-R7, Thermo Scientific), γ-H2AX (1:1000, ab11174, Abcam, Cambridge, UK), and 8-hydroxyguanosine (1:1000, 48508, Abcam, Cambridge, UK), and then processed with the Vectastain Elite ABC Kit (PK6101) and the DAB Peroxidase Substrate Kit (SK4100, Vector Laboratories). Histological slides were scanned with a MIRAX SCAN microscope and processed with the MIRAX Control software (Zeiss). The scans were quantified with Visiopharm software (Visiopharm Integrator System version 2017.2.5.3857).

Cells were seeded on coverslips and LLLT was applied. Forty-eight hours after laser irradiation, cells were fixed with 4% paraformaldehyde acid for 10 min and washed with ice cold methanol at−20°C for 15 min. Ki67 antibody (RM-9106-R7, Thermo Scientific) was incubated for 30 min. Hereafter, cells were incubated with secondary HRP antibodies for 30 min. Cells were analyzed using a light microscope (Olympus). Scoring of Ki67 and mitotic figures was performed in 10 fields with a magnification of 200X by two independent observers.

Cells were fixed by 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.3% Triton X-100 for 15 min at room temperature. After 30-min blocking with 3% BSA, cells were incubated with primary antibodies overnight at 4 °C and on a second day, stained with secondary antibodies. The primary antibodies include rabbit anti-Ki67 (1:1000, RM-9106-R7, Thermo) and anti-ATG7 (1:1000, A7360, Abclonal). DAPI was used as counter staining for nuclei. The intensity of the ATG7 staining was semi-quantified by ImageJ.