Mouse anti ki 67

ChR2-GFP-V6.5 ESCs seeded on Menzel-glass coverslips were fixed by 4% paraformaldehyde for 30 min and washed three times with PBS (5 min per wash). Then, the cells were permeabilized with 0.3% Triton and blocked with 10% bovine serum albumin (BSA) at 37 °C for 60 min and then covered with primary antibody solution and transferred to a cold room at 4 °C overnight. The cells were washed three times, and secondary goat anti-mouse antibody conjugated to Alexa Fluor 555 (Molecular Probe, USA) was added for incubation at 37 °C for 60 min. After the cells were washed three times with PBS, 4′,6-diamidino-2-phenylindole (DAPI) was added for 10 min, and the cells were washed in water three times and mounted using Fluoromount (Dako, Denmark). The primary antibodies were mouse anti-OCT4 (Chemicon, 1:200), Rabbit-anti SOX2 (Abcam, 1:200), Rabbit-anti NANOG (Abcam, 1:200), Mouse-anti Ki67 (Abcam, 1:200) and mouse anti-SSEA1 (Chemicon, 1:200). The images were acquired with a Leica TCS SP2 inverted confocal microscopy system with an HCX PL APO CS 40× 1.25 NA oil immersion objective (Leica, USA). Immunostaining semi-quantification between non-light and light stimulation groups was done as previous [19 (link)].

Slides with sectioned spheroids were rehydrated and washed three times with PBST (PBS with 0.1% Triton X-100). The slides were then incubated in blocking solution containing 5% donkey serum for 1 h, followed by incubation with primary antibodies diluted with the blocking buffer overnight at 4 °C. Slides were washed three times in PBST for 10 min each and incubated with secondary antibodies diluted in blocking buffer for 1 h at room temperature, washed three more times for 10 min each, and cover slipped with ProLong^TM Gold Antifade Mountant with DAPI (cat. P36931, ThermoFisher, Waltham, MA, USA).
The following antibodies were used: rabbit anti-CC3 (1:750, cat. 9661-s, Cell Signaling, Danvers, MA, USA); rat anti-GFAP (1:1000, cat. 13-0300, ThermoFisher); rat anti-CTIP (1:500, cat. ab18465, Abcam, Cambridge, UK); mouse anti-SATB2 (1:100, cat. ab51502, Abcam); rabbit anti-TBR1 (1:250, cat. Ab31940, Abcam); mouse anti-Ki67 (1:50, cat. 550609, BD Pharmingen, San Diego, CA, USA); rabbit anti-MAP2 (1:1000, cat. PA5-85755, Life Technologies); goat anti-Sox2 (cat. AF2018, R&D Systems) mouse anti-TUJ1 (1:250, cat. ab14545, Abcam); mouse anti-CC1 (1:150, cat. ab16794, Abcam) and 4G8 (1:1000, cat. 800701, Biolegend, San Diego, CA, USA) and PHF13 (1:200, cat. 829001, Biolegend). All secondary antibodies were Alexa Fluor conjugated (Life Technologies) and were used at the concentration 1:500.

Immunohistochemistry was performed as described elsewhere [37 (link)]. The following primary antibodies were used for immunohistochemical analysis: mouse anti-Ki-67 (Abcam, Cambridge, UK) and rabbit anti Rig-G (a gift from Dr. Jianhua Tong [7 (link)]). Secondary antibody incubation and staining were performed using the EnVision®+ System–HRP (DAB) kit (Dako), according to the manufacturer's recommendations. TUNEL staining was performed using the DeadEnd Colorimetric TUNEL System kit (Promega, Madison, WI, USA). The number of Ki-67-positive tumor cells and the total number of tumor cells were counted in six microscopic fields of a randomly selected tumor, and the mean value was calculated as the percentage of Ki-67-positive tumor cells.

Cultured cells were fixed in 4% paraformaldehyde for 10 min, then blocked with 10% normal goat serum (NGS) diluted in PBS with 0.3% Triton X-100. Primary antibodies were diluted in 10% NGS in PBS with 0.3% Triton X-100 and incubated in a humid chamber at 4 °C overnight. Secondary antibodies were diluted in PBST and incubated for 1 h at room temperature. Hoechst 33258 was diluted in PBS. The culture was washed three times for 5 min with PBS between each step. The primary antibodies used were mouse anti-βIII-tubulin (1:1000; Covance, Princeton, NJ, USA), rabbit anti-Pax-6 (1:2000; BioLegend, San Diego, CA, USA), rabbit anti-Sox2 (1:100; Millipore, Burlington, MA, USA), mouse anti-Ki67 (1:400; Abcam, Cambridge, UK), and rabbit anti-Cleaved Caspase-3 (CC3) (1:400; Cell Signalling Tech., Danvers, MA, USA). The secondary antibodies used were Alexa Fluor 555- and Alexa Fluor 488-conjugated goat antibodies (1:500; Life Technologies, Carlsbad, CA, USA). Nuclear staining was performed with Hoechst 33343 (1:1000; Sigma, St. Louis, MO, USA). After rinsing with PBS, the coverslips were mounted in a Lab Vision PermaFluor Aqueous Mounting Medium (Thermo Fisher, Waltham, MA, USA). All experiments were repeated at least three times.