All immunostaining was performed after imaging studies with
organoids maintained within the devices, and all reagents were delivered via
microfluidic lines. After fixing and blocking, organoids were stained for
Cadherin-3 (ThermoFisher), Collagen 17 (ThermoFisher), activated Integrin
β1 (9EG7; BD Biosciences), mouse Laminin 332 (gift from Dr. Takako
Sasaki, OITA University), or human Laminin5 γ2 (EMD Millipore); all
primary antibody staining was incubated overnight at 4°C.
Species-specific secondary antibodies (488, 555, or 633 wavelength) and
nuclei staining (DAPI) were also used. Imaging was performed via confocal
microscopy (Zeiss, 63X).
Tumor tissues were fixed with 10% formalin for 24 h, embedded in
paraffin and sectioned (5 mm). After dewaxing using a graded alcohol series
and antigen retrieval, the tumor slides were treated with 3% hydrogen
peroxide, and then blocked with 10% normal goat serum for 1 h. The slides
were incubated with the primary antibodies against Laminin-α3 (1:500)
at 4°C overnight. After three washes with PBS, the tissue sections
were incubated with goat anti-rabbit AlexaFluor 594 antibody (Invitrogen)
for 2h at 4°C. Slides were then mounted using VECTASHIELD (Vector
Laboratories) with DAPI and coverslips applied.
organoids maintained within the devices, and all reagents were delivered via
microfluidic lines. After fixing and blocking, organoids were stained for
Cadherin-3 (ThermoFisher), Collagen 17 (ThermoFisher), activated Integrin
β1 (9EG7; BD Biosciences), mouse Laminin 332 (gift from Dr. Takako
Sasaki, OITA University), or human Laminin5 γ2 (EMD Millipore); all
primary antibody staining was incubated overnight at 4°C.
Species-specific secondary antibodies (488, 555, or 633 wavelength) and
nuclei staining (DAPI) were also used. Imaging was performed via confocal
microscopy (Zeiss, 63X).
Tumor tissues were fixed with 10% formalin for 24 h, embedded in
paraffin and sectioned (5 mm). After dewaxing using a graded alcohol series
and antigen retrieval, the tumor slides were treated with 3% hydrogen
peroxide, and then blocked with 10% normal goat serum for 1 h. The slides
were incubated with the primary antibodies against Laminin-α3 (1:500)
at 4°C overnight. After three washes with PBS, the tissue sections
were incubated with goat anti-rabbit AlexaFluor 594 antibody (Invitrogen)
for 2h at 4°C. Slides were then mounted using VECTASHIELD (Vector
Laboratories) with DAPI and coverslips applied.