Leica dmi8 microscope

Cells were seeded into glass Lab-Tek Chamber Slides (8 wells; 0.8cm2/well) at a density of 15–20 × 10³/well, allowed to attach for 24 h at 37 °C. For P-gp detection AS cells were previously treated with 1 µg/ml docetaxel, 150 µM propranolol and their combination. Once the confluence was reached the cells were fixed for 10 min in acetone at RT and treated with a blocking solution. Then, the slides were incubated with diluted primary antibody anti-β1 Adrenergic Receptor (Abcam), anti-β2 Adrenergic Receptor (Abcam), anti-β3 Adrenergic Receptor (Abcam) and anti-P-gp (JSB-1, Abcam) in 1% BSA in PBS for 1 h at RT. After 3 wash steps with PBS, cells were then incubated with EnVision + System-HRP Labelled polymer secondary antibody (Dako, Denmark). Color development was obtained with AEC ready to use solution (Dako, Denmark), whereas the nuclei were counterstained with hematoxylin. Then, the slides were sealed with Kaiser mounting medium for optical observation.
For the evaluation of CD-31 and CD-34,the slides were incubated with diluted primary antibody anti-CD-31 (PECAM-1, Novocastra) and anti-CD-34 (clone QBEND-10, Novocastra) in 3% BSA in PBS for 1 h and revealed by secondary antibody FITC-Goat anti-mouse Ig (BD Biosciences). Thereafter the slides were sealed with Vectashield (Vector Laboratories) for fluorescence examination by means of DMi8 Leica microscope.

PDMS surface replicas mimicking various textures, placed in the bottom of a polystyrene 24-well chamber, were first sterilized under UV light. PBMCs, pre-stained with CellTracker Fluorescent Probes (Thermo Fisher Scientific Inc.), were seeded onto these surfaces. In experiments involving bacterial interaction, a suspension of S. epidermidis (strain ATCC 12228, OD = 0.1) in Tryptone Broth was added to the wells and incubated for 24 h at 37°C, after which the bacterial medium was replaced with the PBMC solution. Imaging was performed on a DMI8 Leica microscope, utilizing a 20× air objective and maintained under climate control at 37°C. For each experimental condition, 10 images across different vertical planes were captured using an ORCA-Flash 4.0 V3 Digital CMOS camera (Hamamatsu). Time-lapse imaging was conducted at 10-min intervals for up to 5 h, employing Metamorph (v7.10.1.161) for image acquisition.

Tumor tissues were fixed in 10% neutral formalin, embedded in paraffin and cut into 5-μm-thick sections. Immunohistochemistry was performed on a Ventana Discovery Ultra automatic dyeing machine (Ventana Medical Systems, Roche Diagnostics, Indianapolis). The cover glass was loaded on the machine whereupon the sections were dewaxed and rehydrated, followed by endogenous peroxidase activity elimination and antigen retrieval. The sections were incubated with anti-Ki67 antibody (ab16667, Abcam), developed with DISCOVERY ChromoMap DAB Kit and counterstained with hematoxylin (Ventana). The images were collected with a DMi8 Leica microscope and analyzed with Image-Pro Premier software.

Cells were plated immediately after sorting on Matrigel coated chamber slides. 3 hr after plating cells were stained for PAX7 (DSHB). CD56-CD29-CXCR4- cells were used as controls. MYOD and Ki67 protein expression were assessed 3 days after plating. Cryosection slides or sorted human satellite cells were fixed with 4% PFA at room temperature for 10 min, washed in PBST (Phosphate Buffered Saline Tween20 0.1%), permeabilized with 0.1%Triton-100X (Sigma-Aldrich) and then blocked with protein-free serum block (DAKO) or 2% goat serum and incubated at room temperature overnight with primary antibodies (Supplemental Experimental Procedures and Key Resources Table). After PBST wash the corresponding secondary antibodies were applied for 1 hr at room temperature. Sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories) and all samples were examined using a Leica upright or DMi8 Leica microscope.

Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. After permeabilization with 0.3% Triton X-100 (Sigma-Aldrich) in phosphate buffered saline (PBS) for 15 min, the cells were blocked with 10% FBS (Biowest) and 0.1% Triton X-100 in PBS for 1 h at room temperature and then incubated with H1 (1:100, Abcam ab71594) and FAK (1:100, Cell Signaling, 3285) primary antibodies diluted in PBS containing 3% FBS. Goat anti-mouse Alexa-Fluor-647 (A-21235, Life Technologies) and goat anti-rabbit Alexa-Fluor-488 (A-11008, Life Technologies) were used as the secondary antibodies at a concentration of 2 μg/ml in PBS containing 1% FBS for 45 min at room temperature. Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). Slides were mounted with fluorescent mounting medium (Dako Cytomation) and images were acquired with DMi8 Leica microscope. The assays were repeated in three independent biological replicates