Primary antibody of α sma

The mouse lungs were embedded in paraffin, sectioned and subjected to H&E or Masson's trichrome staining. The Ashcroft score was rated and the area of collagen deposition was counted by ImageJ software to quantitatively assess the degree of fibrosis. The range of pathological scoring in Ashcroft score was 0 (normal lung) to 8 (total fibrous obliteration).
For immunohistochemistry analysis, the lung sections were fixed with 4% PFA and the endogenous catalase was removed with methanol containing 3% hydrogen peroxide, followed by recovering the antigen through heating in a pressure cooker with citric acid buffer. The lung tissues were incubated with sheep serum to block the nonspecific sites before incubating with the primary antibody of α-SMA (Abcam) and secondary antibody successively. The stained lung slides were observed with a light microscope.

Immunofluorescence (IF) staining was performed to evaluate the α-SMA expression of the tumor tissues of PDAC patients and the tumor tissue from a PDAC xenograft mouse model. Standard procedures for IF were followed. A primary antibody of α-SMA (Abcam) was diluted 1:100 and incubated with the tissue samples at 4°C overnight. A secondary antibody was added to the samples and incubated for 1 h at room temperature. The sections were then stained with DAPI for 1 min. Sections were observed under a fluorescence microscope.
Sirius red staining was conducted to evaluate the collagen expression of the tumor tissue from the PDAC xenograft mouse model. Sections were deparaffinized and rehydrated, followed by staining with Picrosirius red for 1 h and washing with acidified water twice. The sections were then dehydrated in three changes of 100% ethanol and cleared with xylene. Finally, the sections were mounted in resinous medium and observed under a microscope with a bright field. The scoring for both α-SMA and collagen was based on the principle: 4 for >80% positive cells, 3 for 55%–80% positive cells, 2 for 30%–55% positive cells, and 1 for <30% positive cells.