Cxcl2 FISH and costaining for protein.

Detection of Cxcl2 mRNA and CK-19 or α-SMA protein in formalin-fixed, paraffin-embedded mouse liver tissue was performed using an in situ mRNA and protein costaining protocol described previously (58). Briefly, tissues were deparaffinized, rehydrated, and subjected to heat-mediated antigen retrieval in antigen unmasking solution (Vector Labs). Slides were subsequently incubated in prehybridization solution (3% BSA in 4× SSX) for 20 minutes at 54°C. Tissues were then incubated for 1 hour at 54°C with a fluorescein-labeled Cxcl2 probe (QIAGEN) diluted to 25 nM in hybridization buffer (10% dextran sulfate in 4× SSC). Slides were washed and subjected to a tyramide signal amplification step (PerkinElmer). Slides were washed again, blocked in 3% BSA, and incubated overnight at 4°C with a primary antibody to CK-19 (Abcam) or α-SMA (Abcam). After overnight incubation, slides were washed and incubated with a secondary antibody (Alexa Fluor 594, Thermo Fisher Scientific), washed again, and mounted in ProLong Gold with DAPI (Thermo Fisher Scientific). Slides were analyzed on a Zeiss 710 Confocal Microscope.

Additional information is available in Supplemental Methods.