Alexa fluor 594 goat anti mouse igg

Ki67 primary antibody (Thermo Fisher Scientific, MA5-14520), myosin heavy chain (MHC) primary antibody (Sigma, M4276), skeletal muscle alpha-actin (ACTA1) primary antibody (Sigma, A5228), vascular endothelial growth factor A (VEGFA) primary antibody (Abcam, ab1316), and vimentin primary antibody (Sigma, V6630) were used. Alexa Fluor 594 goat anti-mouse IgG (Biolegend, 405326) was used to detect the primary antibody. Briefly, after antigen retrieval (citrate buffer pH = 6, temperatures below the boiling point, 10 min) and washing (PBS three times), a 15-min incubation with H₂O₂ 10% diluted in methanol was performed. After washing (PBS three times), blocking in goat serum 10% (Sigma, USA) for 30 min at 37 °C was performed. After incubation of the samples with the primary antibody (24 h at 4 °C, humidified environment) and washing (PBS three times), the samples were incubated (2 h in the dark, 37 °C, humidified environment) with the secondary antibody. Finally, DAPI staining was performed to stain the cell nuclei. The slides were photographed using a fluorescence microscope equipped with a camera (× 10).

HT1080 and DF cells were cultured for 48 h inside collagen hydrogels prepared in 12 well glass bottom plates (Cellvis) or inside fibrin hydrogels prepared in 35 mm glass bottom dishes (MatTek). Cells were then fixed in 4% paraformaldehyde (PFA, VWR) for 10 min at room temperature (RT), washed three times with PBS and permeabilized using 0.3% Triton X-100 (Sigma) diluted in PBS. Afterwards, cells were incubated in anti-YAP1 (G6) antibody (sc-376830, Santa Cruz Biotechnologies) for 1 h at RT, washed with 2% bovine serum albumin (BSA, VWR) diluted in PBS and incubated with secondary Alexa Fluor 594 Goat anti-Mouse IgG (BioLegend) antibody for another hour at RT. Subsequently, cells were washed with BSA, incubated with anti-fibronectin antibody (F3648, Sigma) for 1 h at RT, washed again with 2% BSA and incubated with secondary Cyanine5 Goat anti-Rabbit IgG (Invitrogen) antibody for 1 h at RT. Nuclei and F-actin were stained using NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342, Invitrogen) and Alexa Fluor 488 Phalloidin (Invitrogen) diluted in 2% BSA in PBS for 30 min at RT, respectively. Lastly, hydrogels were washed with PBS and covered with Fluoromount-G (Invitrogen).

Jurkat cells were treated with 10 ng/ml TRAIL for apoptosis to occur, following incubation with 10 μM FITC-p17 at 37 °C for 30 min, and then washing with cold PBS two times. After fixation with 100% methanol at −20 °C for 10 min, the cells were permeabilized and blocked in 1% bovine serum albumin in 0.1% PBS-Tween for 1 h at room temperature. Approximately 1 × 10⁶ cells were stained with 5 μl anti-hsp60 antibody (ab13532; Abcam) in 100 μl 1% bovine serum albumin in 0.1% PBS-Tween for 1 h at room temperature. The secondary antibody, Alexa Fluor 594 goat anti-mouse IgG (405326; BioLegend, San Diego, CA, USA), was used at 2.5 μg/ml for 1 h. Resulting cells were mounted using an aqueous mounting medium. Images were taken using the Olympus FV1000 Inverted Confocal IX81 Microscope (FV1000+IX81; Olympus America Inc.) and analyzed by FV10-ASW software (Olympus) at an excitation wavelength of 559 nm for Alexa Fluor 549 and 488 nm for FITC. The colocalization of merged images was analyzed by Image-Pro Plus (Media Cybernetics, Rockville, MD, USA).