Ab254357

BMDMs and THP-1 cells were treated with IL-4 (MUS: Pepro Tech, 214–14; HUM: Pepro Tech, 200–04) or a 1:1 solution of PDAC-CM and complete medium containing 10% FBS for 48 h. NuPAGE™ LDS Sample Buffer (Invitrogen, NP0007) was directly added to the cell culture plate to lyse the cells on ice. The protein was heated in an iron bath at 70°C for 10 min, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and electrotransferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore). The main antibodies used in the research were CD206 (ab64693, Abcam), Arg-1 (93,668, CST), p-Akt (4060S, CST), Akt (4691S, CST), p-Stat6 (ab263947, Abcam), Stat6 (ab32520, Abcam), p-mTOR (5,536, CST), mTOR (2,983, CST), mouse IL4Rα (MAB530, R&D system), human IL4Rα (MAB230, R&D system), CSF1R (ab254357, Abcam), p-Stat3 (ab76315, Abcam), and Ym-1 (ab192029, Abcam). The protein bands were observed using an enhanced chemiluminescence (ECL) detection kit (P10100, NCM), and images were obtained using a chemiluminescence detection system (Bio–Rad).

All embryos were fixed overnight in 4% paraformaldehyde, dehydrated through graded alcohols, embedded in paraffin, and sectioned at a thickness of 4 µm. For TRAP staining, sections were incubated with pure water at 37 °C for 2 h, placed in the prepared TRAP staining solution at 37 °C for dark staining for 1 h, and then counterstained with haematoxylin. For von Kossa staining, the sections were dropped with the prepared von Kossa silver staining solution and exposed to bright light for 15 min, followed by Hywave solution for 2 min, and finally counterstained with eosin. A TRAP kit (387A-1KT, Sigma, USA) and von Kossa kit (G3282, Solarbio, China) were used as described in our previous study^{9 (link)}. For immunohistochemistry, sections were subjected to antigen retrieval and then blocked with normal goat serum, followed by incubation overnight at 4 °C with the primary antibodies anti-CD14 (1:200, ab181470, Abcam), anti-F4/80 (1:200, ab300421, Abcam), anti-CSF1R (1:200, ab254357, Abcam), and anti-RANKL (1:200, ab45039, Abcam). Anti-CD31 (1:300, ab28364, Abcam) and goat anti-rabbit IgG H&L (1:200, ab150080, Abcam) were used.