Sc 9974

Tissues were rinsed with 1 × PBS thrice at room temperature. Blocking was done with 1% BSA (NEB) and 0.1% Tween-20 in 1 × PBS for 1 h at room temperature. Tissues were stained at 4 °C overnight using the following antibodies diluted in blocking solution: anti-LUM (Abcam, ab168384; clone EPR11380(B); Lot GR121948-4; 1:75), anti-MMP2 (Abcam, ab97779; Lot GR3448382-1; 1:200), anti-α-SMA (Abcam, ab7817; clone 1A4; Lot 1009584-11;1:600), and anti-PDGFA (Santa Cruz Biotechnology, sc-9974; clone E-10; Lot C0222; 1:600). PDPN was detected using AF488-conjugated primary antibody (BioLegend, 337005; clone NC-08; Lot B360564; 1:75). Secondary antibody staining was then carried out for 1 h at room temperate using anti-mouse AF594 (ThermoFisher, A11005; Lot 2538976; 1:1000) and anti-rabbit AF488 (ThermoFisher, A11008; Lot 2557379; 1:1000). Finally, samples were stained with anti-CD68 (Cell Signaling Technology, #79594; clone D4B9C; Lot 779594S; 1:50) overnight at 4 °C. After washing with 1 × PBS three times, tissues were counterstained with DAPI (Sigma) before mounting (Vectashield, H-1700-10).

The tissue microarrays were purchased from Biomax (GL481, Rockville, MD, USA). IHC staining was performed using a Ventana BenchMark XT automated stainer (Ventana, Tucson, AZ, USA) and specific primary antibodies against CEBPD (ab65081, Cambridge, MA, USA) and PDGFA (sc-9974, Santa Cruz, CA, USA). The H-scores ranging from 0 to 300 were calculated by multiplying the staining intensity by the percentage of each core.

Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tumor samples sliced at 4 μm. Samples were stained for CD68 (1:200; MCA1957, BioRad, Hercules, CA, USA), and PDGF-AA (1:250; sc-9974, Santa Cruz Biotechnology, Dallas, TX, USA) following protocol by Pérez-Morales et al., 2018 [48 (link)]. Briefly, slides were heated at 65 °C for 30 min and then deparaffinized using graded xylenes. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 15 min. Antigen retrieval was performed using citrate buffer solution for 40 min in a preheated water bath (95–99 °C) and followed by a 30-min cool-down. Protein block solution was added for one hrs at room temperature. Tissues were incubated overnight in the primary antibody at 4–8 °C in a humidity chamber. Protein block, secondary antibody, and 3,3′-Diaminobenzidine (DAB) in the Super Sensitive Link Label IHC kit (LP000-ULE, BioGenex, Fremont, CA, USA) were used for visualization per manufacturer’s instructions. Counterstain was performed using hematoxylin for 20 s, followed by washing with running tap water for five minutes. Slides were dried, mounted, and then blindly scored by two independent investigators. CD68 counts were determined by the amount of CD68+ cells per high power field (HPF) using ImageJ software (Bethesda, MD, USA).