Ab192029

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).

Immunofluorescence for F4/80, iNOS, and YM1 was performed by incubating the sections with Alexa Fluor 594 (1 : 300, ThermoFisher, #A11007) anti-mouse and Alexa Fluor 488 anti-rabbit (1 : 300, ThermoFisher, #A11034) anti-mouse sections. The nuclei were stained with DAPI (1 : 600, ThermoFisher, #D1306). The kidney slices were incubated with primary mouse anti-F4/80 antibodies (1 : 500, Abcam, #ab6640) overnight at 4°C, rabbit anti-iNOS (1 : 100, Abcam, #ab15323), and rabbit anti-YM-1+YM-2, (1 : 10000, Abcam, #ab192029). Nonspecific binding was controlled by the replacement of a negative control by the primary antibody.

The 4% paraformaldehyde fixed cells for 10 min at room temperature. Next, cells were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. After that, the antibody of Ym1/2 (ab192029, 1:100; Abcam, UK) and Cox2 (ab179800, 1:100; Abcam, UK) or β-tubulin III (ab18207, 1:2,000: Abcam, UK) was used to incubate the cells for 12 h at 4°C. Next, the cells were washed three times with PBS. Next, the Cy3-conjugated AffiniPure IgG (HL) secondary antibody (1:800, Abcam, UK) was used to treat cells for 1 h in the dark and was washed three times with PBS. Next, the sections were stained by 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and were detected using an Afv10i confocal microscope (Keyence, Japan) at 495, 565, and 400 nm. The expressions of COX2 and Ym1/2, the axon length, and neurite branches of neuronal cells were quantitatively analyzed using ImageJ (ImageJ 1.48v, NIH).

Total proteins were extracted from the indicated tissues and cells by using a protein extraction kit (Pierce Biotechnology Inc., IL, USA) in accordance with the manufacturer’s protocol. The collected proteins were loaded and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and then transferred onto polyvinylidene fluoride (PVDF) membranes which were rocked gently for at least 1 h in blocking buffer (5% milk in TBST). Subsequently, the membranes were incubated overnight with primary antibodies; namely, iNOS (1:1000, ab15323, Abcam, UK), Ym-1 (1:1000, ab192029, Abcam, UK), CD86 (1:1000, ab167720, Abcam, UK), Arg-1 (1:1000, 89,007, Abcam, UK), MIF (1:1000, ab187064, Abcam, UK), CD74 (1:1000, ab270265, Abcam, UK), ERK1/2 (1:1000, ab184699, Abcam, UK), p-ERK1/2 (1:1000, ab278538, Abcam, UK), AKT (1:1000, ab8805, Abcam, UK), p-AKT (1:1000, ab38449, Abcam, UK),and GAPDH (1:2000, AC002, ABclonal, China). After incubating with horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse secondary antibody (1:5000, BA1054/BA1050, Boster, China) for 1 h, the protein bands were detected with the ECL Western blot detection kit in an enhanced chemiluminescence system.

All tissues or cells were lysed in RIPA buffer, and total protein concentrations were determined with BCA Protein Assay Kit. Total protein was loaded into precast 8%-12% SDS-PAGE gels and then transferred onto PVDF membranes. After that, the membranes were incubated with the following primary antibodies at 4 °C overnight: anti-β-actin (Servicebio, GB11001, 1:1000), anti-YM-1 (Abcam, ab192029, 1:2000), anti-CD206 (Abcam, ab64693, 1:1000), anti-LC3A/B (Cell signalling, #12741, 1:1000), anti-Beclin1 (Abcam, ab207612, 1:1000), anti-ATG5 (Proteintech, 10181-2-AP, 1:1000), anti-p62 (Abcam, ab56416, 1:2000), anti-IL-1β (Abcam, ab9722, 1:1000). The primary antibodies were recycled and membranes were washed 3 times in Tris-buffered saline with Tween-20 (TBST) for 6 min each time. Goat anti-Rabbit IgG and Goat anti-Mouse IgG secondary antibody (Jackson ImmunoResearch, 103069, 1:10,000) were incubated for 1 h at room temperature. After the removal of the secondary antibody, the membranes were washed 3 times with TBST for 6 min each time. Finally, the relative proteins levels were detected by enhanced chemiluminescence reagent and ImageJ software.

Total proteins were extracted from the brain tissues and BV-2 cells by using a protein extraction kit (Pierce Biotechnology Inc., IL, USA) in accordance with the manufacturer’s protocol. Total proteins were loaded and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred onto polyvinylidene fluoride (PVDF) membranes, rock gently for at least 1 h in blocking buffer (5% milk in TBST). Subsequently, membranes were incubated overnight with primary antibodies; namely, iNOS (1:1000, ab15323, Abcam, UK), Ym-1 (1:1000, ab192029, Abcam, UK), CD86 (1:1000, ab167720, Abcam, UK), Arg-1 (1:1000, 89007, Abcam, UK), CX3CR1 (1:500, ab8021, Abcam, UK), and GAPDH (1:2000, AC002, ABclonal, China). Next, membranes were incubated with horse-radish peroxidase labeled goat anti-rabbit or goat anti-mouse secondary antibody (1:5000, BA1054/BA1050, Boster, China) for 1 h. The ECL Western blot detection kit and an enhanced chemiluminescence system were used to examine the protein bands. The experiment was repeated three times and all measured protein levels were quantified by densitometry.

Four-micron-thick plaque sample sections were incubated with a Total galactose-specific lectin 3 (MAC-2) antibody (Abcam, ab2785, 1:100), total chitinase 3-like 3 (YM-1) antibody (Abcam, ab192029, 1:500), AP-1 antibody (Abcam, ab40766, 1:100) or the proliferating cell nuclear antigen (PCNA) antibody (Abcam, ab29, 1:100) overnight at 4 °C after blocking with 1% goat serum. Slides underwent color development with DAB and hematoxylin counterstaining. Images were obtained with an optical microscope (Leica DMI6000B).