Tsa green kit

Immunohistochemistry/immunofluorescence was performed as follows: cryosections were incubated with blocking buffer (5% goat serum, 5% donkey serum, 0.5% BSA, 0.25% Triton-X 100 in PBS) for 1 h and labeled with primary antibodies (Supplementary Table S2) or isotype controls overnight at 4°C in blocking buffer. The following day, sections were washed three times with washing buffer (0.2% Tween-20 in PBS) and incubated with fluorescence probe-conjugated secondary antibodies for 1 h at room temperature. We used mannose receptor-1 (CD206) as a marker for resident macrophages and platelet-derived growth factor receptor α (PDGFRα) as a marker for FAPs. The TSA Green kit (Tyramide Signal Amplification; Perkin Elmer, Waltham, MA) was used for CD206 and PFGFRα staining after 1 h of incubation with biotinylated donkey-anti-rabbit F(ab′)2 IgG fragments (2.5 µg/ml) to enhance the immunostaining signal. Nuclei were then stained with DAPI (1 µg/ml) and mounted using Vectashield (Vector Labs, Burlingame, CA). All images were taken on a Revolve Echo widefield fluorescence microscope using a x10 (PlanC N, Olympus) or a x20 objective (UPlanFL N, Olympus) and 5 MP CMOS Monochrome Camera.

IF was performed as follows: cells were fixed with 4% paraformaldehyde (PFA) for 10 min, incubated with blocking buffer (5% goat serum, 0.5% BSA, 0.25% Triton-X 100 in PBS) for 1 h, and labeled with primary antibodies (shown in Additional file 4.) overnight at 4 ˚C. The following day, cells were washed thrice with washing buffer (0.2% Tween-20 and PBS) and incubated with the appropriate secondary antibodies for 1 h at room temperature. For cytokeratin 7 (KRT7) imaging, after a 1-h incubation with biotinylated goat-anti-mouse F(ab’)2 IgG fragments (2.5 µg/mL), a TSA Green kit (Tyramide Signal Amplification; Perkin Elmer, www.perkinelmer.com, Waltham, MA) was used to enhance the immunostaining signal. Nuclei were stained with Hoechst 33,342 and mounted on Vectashield (Vector Labs, www.vectorlabs.com, Burlingame, CA). Stained cells were examined using a confocal microscope and the ZEN 2009 Light Edition software (Carl Zeiss, Oberkochen, Germany).