Fluorescein isothiocyanate (fitc)

For paraffin tissue sections, sections were dewaxed with xylene and rehydrated with an alcohol series. Antigen retrieval was performed by heating the slides at 95°C for 25 minutes in a working solution of Target Retrieval Solution (DAKO). The slides were permeabilized with phosphate-buffered saline with 0.01% Triton for 10 minutes. Slides were blocked with 4% bovine serum albumen in phosphate-buffered saline with 0.01% Triton for 10 minutes. To detect eGFP-expressing human tumor cells in tissue sections, primary goat antibodies (against eGFP) directly conjugated to fluorescein isothiocyanate (Abcam) were used. To detect GRP78, polyclonal rabbit anti-GRP78 antibodies (1:100, Abcam) were used. Goat anti-rabbit secondary antibodies conjugated to Alexa 594 were used to localize rabbit anti-GRP78 in tissue sections. Nuclei were counterstained blue with 4′,6-diamidino-2-phenylindole (DAPI, 1 mg/ml; Sigma) for 5 minutes. Tissue sections stained only with secondary antibodies served as omission controls to assess the level of background fluorescence. Tissue sections were imaged using a Zeiss 710 laser scanning confocal microscope equipped with argon, DPSS 561, and Diode 405 lasers, where images were acquired using either a low-power (20×) objective lens or 63× oil immersion objective lens.

Cells were exposed to X-ray radiation (1 Gy). Then, the cells were fixed with 4% paraformaldehyde at 0.5, 4 and 24 h, permeabilized with 0.1% Triton X-100, stained with γ-H2AX antibody (Rabbit IgG, Cat. # ab11174, Abcam) (diluted 1:200) at 4°C overnight and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Goat anti-Rabbit IgG, Cat. # 65–6111, Thermo Fisher Scientific) (diluted 1:100) for 1 h at room temperature. Finally, the slides were observed under a laser scanning confocal microscope (Zeiss LSM510).

The attachment and proliferation of MC3T3-E1 cells on the specimens was investigated by utilizing methylthiazol tetrazolium test (MTT) at different time after culturing.20 (link),21 (link) After the specimens were placed into the plates, 0.1 mL of the MTT solution (0.5 mg/mL) was added into the plates, and the specimens were incubated at 37°C with 5% CO₂ for 4 hours. After incubation, 500 μL dimethyl sulfoxide (Sigma-Aldrich Co., USA) was added into the plates to dissolve the purple formazan, and then mixed by pipetting the solution. Then, the solution was incubated for 10 minutes (at 37°C). The optical density (OD) values of MC3T3-E1 cells on specimens were determined at 570 nm by using automated plate reader (Varioskan LUX, Thermo Scientific Co., USA). Furthermore, the specimens were separately stained by utilizing fluorescein isothiocyanate (Abcam Co., UK) and 4ʹ,6ʹ-diamidino-2-phenylindole (Abcam Co., UK) for 40 minutes and 5 minutes, and the cell morphology of MC3T3-E1 cells on the specimens at 24 hours was observed by using confocal laser scanning microscope (CLSM; Nikon A1R, Nikon Co., Japan).

4-dimethylaminocinnamaldehyde (DMAC), diphenylpicrylhydrazyl (DPPH), 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and propidium iodide (PI) were purchased from Sigma Aldrich, USA. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from HiMedia Laboratories Pvt. Ltd., France. Gallic acid was purchased from Loba Chemie Pvt. Ltd., India, (+) catechin from Natural Remedies Pvt. Ltd, India, and procyanidin B2 from Santa Cruz Biotechnology, USA. Doxorubicin hydrochloride API was a generous gift from Veeda Clinical Research Pvt. Ltd., India. Cell culture media and reagents were purchased from Genetix Biotech Asia Pvt. Ltd., India. P-gp monoclonal antibody (clone UIC2) and IgG2a isotype control, conjugated with fluorescein isothiocyanate (FITC), were purchased from Abcam, UK. All the other analytical chemicals and solvents were purchased from Fisher Scientific, USA.