Tcs sp5 microsystems

After completion of imaging, the animals were euthanized, and the colon was excised, flushed with PBS, and divided longitudinally for macroscopic imaging (IVIS 200, Caliper Life Sciences), to validate specific uptake by the EGFR peptide. NIR fluorescence images were collected using a Cy5.5 filter with λ_ex = 675 nm excitation and 720 nm emission with 0.05 sec exposure. A ruler was placed next to the specimen to determine the distance from the anus for registration with the endoscopy and histology images. Whole mount tissue was imaged with a conventional tabletop confocal microscope (Leica TCS SP5 Microsystems) using Cy5.5 and DAPI filters. The colon was then fixed in 10% buffered formalin and processed for routine histology (H&E).

HeLa cells were cultured on a Lab-Tek Chamber glass slide. Fluo-4-AM (5 μM), MitoSOX (5 μM), and DCF (5 μM) (Thermo Fisher, MA, USA) were added in HBSS buffer (0.49 mM MgCl₂, 0.41 mM MgSO₄, 5.33 mM KCl, 0.44 mM KH₂PO₄, 4.17 mM NaHCO₃, 137.93 mM NaCl, 0.34 mM Na₂HPO₄, 5.56 mM D-glucose, with or without 1.26 mM CaCl₂) (Gibco-Thermo Fisher, MA, USA) and incubated for 10 min. Afterward, the cells were washed once with HBSS, and peptides were treated with HBSS. Time-lapse images were obtained using an Argon laser scanning confocal microscope (Leica TCS SP5 Microsystems, Wetzlar, Germany) at 10-s intervals for 10 min at an excitation of 488 nm for Fluo-4 and MitoSOX, or at 496 nm for DCF. As mitochondrial markers, MitoTracker Green (0.5 μM) and MitoTracker Red (0.1 μM) were preincubated (green for 30 min and red for 2 min), anti-TOMM20 antibody (Abcam, Cambridge, UK) was used for immunocytochemistry and immunofluorescence (ICC/IF), and mito-DsRed2 were transfected with Effectene (Qiagen, Hilden, Germany) on the day before confocal microscopy. Other details are available in the manufacturer’s instructions.

For phalloidin staining, 1.5 × 10⁴ cells per well were seeded in a 15-mm glass bottom cell culture dish and treated with tyrosol as described above. Cell fixation was done at room temperature using 4% paraformaldehyde for 30 min. Cells were then permeabilized with 0.1% Triton X-100 diluted with PBS for 5 min, followed by blocking using 1% bovine serum albumin for 1 h. Phalloidin staining of the cells was done by incubating the samples at 37°C for 60 min with phalloidin. Images were captured with Microsystems-TCS SP5 (Leica). Results are shown as fractal dimension quantification, representing the G-actin polymerization formed from F-actin. Quantification analysis was performed using ImageJ software as described previously (Vince et al., 2008 (link)).

Frozen gastrocnemius muscles were sectioned at 10 μm thickness using a cryostat and subjected to immunohistochemistry as described above [14 (link)].
For Ki67 staining, cells cultured under indicated condition and treated with salidroside or PBS were seeded in a 15-mm glass bottom cell culture dish, and then further cultured for 12 h. Cells were fixed with 4% paraformaldehyde and permeabilized for 5 min with PBS containing 0.1% Triton X-100. After blocking with 1% bovine serum albumin for 1 h, the samples were incubated at room temperature for 90 min with an anti-Ki67 antibody and subsequently, stained with secondary antibodies anti-rabbit Alexa Fluor 488 (Invitrogen Life Technologies). DAPI (Beyotime, Guangzhou, China) was used to stain nuclei. Images were taken with Microsystems-AF6000 (Leica, Heidelberg, Germany). For phalloidin staining, samples were incubated at room temperature for 30 min with phalloidin after blocking, and images were taken with Microsystems-TCS SP5 (Leica). The quantification of F-Actin formed from G-Actin polymerization was performed by fractal dimension as described previously [25 (link)], by using ImageJ software. For experiments with conditioned media, the cells were cultured with conditioned medium under hypoxia for 12 h prior to staining. The antibodies used were listed in Supplementary Table 2.

Clinical human breast carcinoma tissues were excised, freezed and sectioned into 10 μm slices using a cryostat. Tissue sections were incubated with a primary antibody for 1 h prior to staining with secondary antibodies conjugated with anti-rabbit Alexa Fluor 488 or anti-mouse Alexa Fluor 568 (Invitrogen Life Technologies). For Ki67 staining, cells transfected with indicated shRNA expression vectors and selected by using puromycin were re-seeded in a 3.5 mm cell culture dish. Cells were fixed with 4% paraformaldehide and permeabilized for 30 min with PBS containing 0.1% Triton X-100. After blocking with 2.5% bovine serum, the cells were incubated at room temperature for 1 h with primary antibody, followed by staining with secondary antibodies (anti-rabbit Alexa Fluor 488, Invitrogen Life Technologies). 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Guangzhou, China) were used to stain nuclei. Images were taken with Microsystems-TCS SP5 (Leica, Heidelberg, Germany). The antibodies used are shown in Supplementary Table 2.