Ab9693

The reconstitution solution (batch ID APL-0917-5991-03), was produced by Aesica Pharmaceuticals, UK, for the reconstitution of the AUP1602-C and was also used in the studies as vehicle. The reconstitution solution is a benign hypertonic solution containing 5% dextrose, 2.5% saline and 1.6% sodium acetate (pH 6.0–6.5).
Antibodies used in the western blotting and immunohistochemical stainings were rabbit anti-FGF2 (ab246354, 1:70, Abcam, UK), rabbit anti-IL-4 (ab9622, 1:70, Abcam, UK), rabbit anti-CSF-1 (ab9693, 1:80, Abcam, UK), rat anti-neutrophil NIMP-14 (ab2577, 1:100, Abcam, UK), rat anti-macrophage F4/80 (ab16911, 1:200, Abcam, UK), rabbit anti-CD31 (ab28364, 1:50, Abcam, UK), rat anti-BrdU (ab6326, 1:500, Abcam, UK).

Western blot analysis was performed as described previously^{59 (link)}. Briefly, proteins were electrophoresed in 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were blocked in 5% milk and incubated overnight at 4 °C with the primary antibodies, followed by incubation with anti-rabbit Dye 680CW or anti-mouse Dye 800CW (LI-COR, MO, USA) at 1/10,000 dilution for 1 h. The specific signals and the corresponding band intensities were evaluated using Odyssey Infrared Imaging system (Odyssey, Berlin, Germany). The protein level was normalized against GAPDH. The following antibodies were used in this study: rabbit anti-CSF1 (0.2 µg/ml; Abcam, ab9693), rabbit anti-CX3CL1 (1/5,000 dilution; Abcam, ab85034), and mouse anti-GAPDH (1/10,000 dilution; Bioworld, MB001).

Cells were lysed in 200 μl lysis buffer (0.5 M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS, and 5% β-mercaptoethanol). Protein lysates (20 μg) were electrophoresed on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dried milk and incubated overnight at 4 °C with the appropriate antibodies, followed by a horseradish peroxidase-conjugated secondary antibody. The immunocomplexes were visualized using a chemiluminescence phototope–horseradish peroxidase Kit (Cell Signaling Technologies, Danvers, MA, USA). Each primary antibody was validated for the relevant species and applications, as shown on the manufacturers’ websites, including CREBBP (amino acids 162–176, ab2832, Abcam, Cambridge, UK), EP300 (ab14984, Abcam), H3K27ac (ab4729, Abcam), Anti-Flag (F1804, Merck, Kenilworth, NJ, USA), NICD (ab8925, Abcam), FBXW7 (ab109617, Abcam), CCL2 (66272, Proteintech, Chicago, IL, USA) and CSF1 (ab9693, Abcam). Tubulin (ab7291, Abcam), and H3 (17168, Proteintech) were used to ensure equivalent loading of protein. Horseradish peroxidase-conjugated secondary antibodies against goat anti-mouse-IgG and goat anti-rabbit-IgG were from Cell Signaling Technologies. The immunoreactive band intensities were quantified with ImageJ.