Goat anti mouse igg h l cross adsorbed secondary antibody alexa fluor 594

For cell samples, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 PBS solution. After blocking with 5% bovine serum albumin (BSA, Beyotime, >99%), cells were incubated overnight at 4 °C with indicated primary antibodies (γ-H2AX, 1:200, Abcam; cTnT, 1:500, Abcam; β-actin, 1:1000, Proteintech; Alexa Fluor™ Plus 647 Phalloidin, 1:1000, ThermoFisher). Incubation with fluorescently coupled secondary antibodies (Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor™ 594, 1:1000, ThermoFisher; Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor™ 488, 1:1000, ThermoFisher; Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor™ 594, 1:1000, ThermoFisher) was followed by further washing in PBS. After washing, nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI, ThermoFisher). For histological samples, 7 µm tissue cryosections were washed with PBS to remove OCT. After fixation and permeabilization blocking, tissues were stained with the indicated primary antibody overnight. Afterward, the tissue was rinsed, stained with fluorescently coupled secondary antibodies for 1 h at room temperature, and then mounted with DAPI. The slides were visualized using a fluorescence microscope.

Immunocytochemistry was performed using 8-well chamber slides (Nunc™ Lab-Tek™ Chamber Slide System, Thermo Fisher Scientific, Waltham, MA, USA. Briefly, 5000 or 10,000 cells (meningioma and schwannoma, respectively) were plated in each well, and left to grow overnight before adding drug treatments. After the drug treatment, the cells were fixed with 4% paraformaldehyde (Thermo Fisher Scientific)/PBS. Cells were permeabilised with 0.2% Triton X-100 for 5 min; blocking was performed with 10% v/v goat serum (Abcam, Cambridge, UK) in 1% w/v bovine serum albumin (BSA) (Thermo Fisher Scientific)/PBS for 1 h. Cells were incubated overnight with Ki-67 (1:200) (DAKO) in 1% BSA/PBS and then Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific) (1:500) in 1% BSA/PBS for 1 h at room temperature. Finally, 4’,6-diamidino-2-phenylindole (DAPI) nuclear stain was added (1:500) before imaging with an inverted Leica DMi8 microscope. Ki-67 staining was quantified by manually counting the number of Ki-67 positive cells versus the total number of cells (positive for DAPI staining) using ImageJ software. Statistical analysis was performed on raw data using a Repeated Measures ANOVA (with Tukey’s Multiple Comparison Test).

MFF-1 cells were seeded onto coverslips in 24-well plates and were co-transfected with 0.25 µg of each plasmid in same combinations as described above. 24 hours after transfection, the supernatants were removed, and the cells were washed with PBS and fixed by using 4% paraformaldehyde at 28°C for 40 min. Next, the cells were washed four times with PBS and incubated with 0.2% Triton X-100 to permeate the cell membrane at 28°C for 12 min. After being washed with PBS for four times, the cells were blocked by 5% BSA at 37°C for 1 hour and incubated with the tag-antibodies described above and the fluorescein-labelled secondary antibodies, including Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (Thermo Fisher Scientific) or Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Thermo Fisher Scientific). Subsequently, the coverslips with labelled cells were sticked on the microscope slides by using ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). The cells were observed under a Leica confocal microscope (40 × magnification, oil immersion lens).