Masson s trichrome stain kit

Serial sections of human and mouse pancreatic tumor tissues were stained for collagen with the Masson's trichrome stain kit (Sigma-Aldrich) according to the manufacturer's protocol. Hematoxylin was used to counterstain the cell nuclei. Bright-field images were captured using a BX51 microscope (OLYMPUS).

Mice were euthanized and lung tissues were harvested and fixed in 10% (v/v) neutral buffered formalin before preparation of paraffin sections. Paraffin-embedded sections were deparaffinized and stained with hematoxylin and eosin (H&E), using a Masson's trichrome stain kit or Sirius red stain kit (Sigma-Aldrich) to detect collagen.
Before immunohistochemistry, deparaffinized sections were in antigen-retrieval buffer (Abcam) for 30 min and next incubated with 0.3% (v/v) hydrogen peroxide in methanol for 10 min. Sections were blocked in normal horse serum at 37 °C incubator for 1 h and immunostained overnight at 4 °C with primary antibodies. The target proteins were visualized using ABC and DAB kits (Vector Laboratories) and counterstained with hematoxylin.
For immunofluorescence staining, cell or sections were stained with primary antibodies and incubated with Alexa 488- labeled anti-mouse, Alexa 568- labeled anti-rabbit, Alexa 647- labeled anti-goat labeled secondary antibodies (1:1000; Thermofisher) and counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; 3 mmol/L). Images were obtained using a Zeiss microscope Apotome (Cal Zeiss), equipped at Ewha Drug Development Research Core Center. The detailed protocol and antibodies for immunohistochemical and immunofluorescence staining were provided in Additional file 1: Table S2.