Alexa 488 conjugated secondary antibody

Cells were sonoporated with eGFP pDNA in 16well chamber slides and were fixed with 4% paraformaldehyde after 24 h of sonication. eGFP was immune-stained with an anti-GFP monoclonal antibody (Invitrogen, Carlsbad, CA, USA) at a dilution of 1:500, an Alexa488-conjugated secondary antibody (Abcam, Cambridge, MA, USA) and Hoechst 33342 (Dojindo Molecular Technologies, Kumamoto, JP) Fluorescence images of cells were analyzed using BZ-X710 digital microscopy (KEYENCE, Osaka, JP).

After indicated transfection, HSC-3 cells were soaked in paraformaldehyde fixation solution (4%) and permeation agent (containing 0.2% Triton X-100), and sealed in 5% BSA. Subsequently, the cells were treated by primary antibodies that recognize E-cad (cat. no. ab308347; 1/500) and N-cad (cat. no. ab18203; 1/500) from Abcam overnight at 4 °C and Alexa-488-conjugated secondary antibody (cat. no. ab150077; 1/200; Abcam) for 1 h at room temperature. The nuclei were labelled with 5 µg/ml DAPI for 5 min. Images were observed under a fluorescence microscope (Olympus, Tokyo, Japan).

After paraformaldehyde fixation, brains were cut with a cryostate (Leica Microsystems, Buffalo Grove, IL, USA) to obtain 50-μm thick coronal sections of the mesencephalon (~15 sections/brain). Sections were then probed with a mouse monoclonal anti-Tyrosine Hydroxylase (anti-TH) antibody (1:500; Boster Bio, CA, USA) and revealed with an Alexa 488-conjugated secondary antibody (1:500; Abcam, Cambridge, UK). Images were taken with the 10× objective of an epifluorescence microscope (Olympus BX63, Milan, Italy), then digitally reconstructed with the CellSens Dimension software. Quantitative analysis was performed on images of to the ventral half of whole sections according to a previously published method (Gerace et al., 2014 (link)). In brief, mean pixel intensity was measured in same-size square regions of interest from the drug-injected and the saline-injected areas of each brain. This value was then normalized to the total intensity of the entire TH-positive area of the drug-injected side and compared to corresponding value of the contralateral, saline-injected side (×5 sections, each brain).

Tumor tissues embedded in OCT compound (DAKO, Carpinteria, CA, USA) were rapidly frozen in liquid nitrogen and cut using Cryostat Leica CM 3050S microcryotome (Leica, Wetzlar, Germany). The tissue sections were fixed with 16% formaldehyde and stained with hematoxylin and eosin as described previously [43 (link)]. For evaluation of in vivo cell proliferation, the sections were incubated with anti-Ki67 and then stained with Alexa 488-conjugated secondary antibody (Abcam, Cambridge, MA, USA). After counterstaining the sections with DAPI (4, 6-diamidino-2-phenylindole), images were captured with a Olympus IX71 fluorescence microscope (Olympus, Tokyo, Japan). For analysis of apoptosis in the sections, terminal dUTP nick-end labeling (TUNEL) assay was performed using the Promega DeadEnd™ according to manufacturer’s instructions.

SW1573 cells were seeded at 8000 cell/well density on an 8-well chamber slide (Thermo Fisher Scientific, Nunc™ Lab-Tek™ Chamber Slide System, Cat.no: 177402). The next day they were treated with 500 nM tipifarnib, 100 nM sotorasib or their combination for 48 h, then fixed with 4% formaldehyde for 15 min. Cells were permeabilized with 0.1% Triton-X100 for 10 min, washed with DPBS and incubated with lamin A/C primary antibody (4777 T, Cell Signaling) dissolved in DPBS (1:200) for an hour at RT. Cells were repeatedly washed with DPBS and incubated with Alexa488 conjugated secondary antibody (Abcam, ab150113) dissolved in DPBS (1:1000) for half an hour at RT. Cells were washed again with DPBS and covered with Vectashield Antifade Mounting Media containing DAPI. For imaging we used a Leica DM RXA epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with DAPI (excitation 355–425 nm, emission >470 nm, Leica Microsystems, Wetzlar, Germany), Spectrum Green (excitation 460–500 nm, emission 512–542 nm, Vysis, Downers Grove, IL, USA) filter sets for blue and green, respectively. Image acquisition was performed using a Leica DFC365 FX camera and Leica CW4000 FISH software. Two independent experiments were performed for lamin immunofluorescence imaging.

Human recombinant TGF-β2 (R&D systems, Minneapolis, MN) stock solution (20 μg/mL) was prepared with 0.1% BSA/4 mM HCL according to manufacturer’s procedure, and used at a final concentration of 5 ng/mL An ALK inhibitor, SB431542 was obtained from Stemgent (Cambridge, MA). SB-525334 was from Sigma-Aldrich (St. Louis, MO). SU9516 was from Merck Millipore (Billerica, MA). These compound were dissolved in DMSO at 10 mM as a stock solution and diluted when used. Antibodies used were: E-cadherin (mouse monoclonal, clone 36/E-cadherin), obtained from BD Biosciences (San Jose, CA). An anti-β-actin antibody (mouse monoclonal, clone AC-15) was obtained from Sigma–Aldrich (St. Louis, MO). All secondary horseradish peroxidase-conjugated antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Alexa 488-conjugated secondary antibody was obtained from Abcam (Cambridge, MA).