Fig 1: Loss of SOCS3 in muscle stem cells in vivo does not affect the inflammatory response after myotoxic injury.Tamoxifen-treated control (SOCS3fl/fl Pax7-CreER-) and SOCS3 MKO (SOCS3fl/fl Pax7-CreER+) mice were left uninjured (UN) or received a 40 µL injection of notexin (10 µg/ml) into the right TA muscle. Mice were killed for analysis at 1 (D1), 2 (D2), 3 (D3), 7 (D7) or 14 days (D14) post-notexin injury. Protein was extracted from right TA muscles after sectioning and western immunoblotting for phosphorylated and total STAT3 protein. (A) Representative immunoblots for phosphorylated (top) and total (bottom) STAT3 protein levels are shown. Band intensity was quantified using ImageQuant software (Bio-Rad Laboratories) and the ratio of phosphorylated/total STAT3 protein levels was determined. RNA was extracted from snap frozen muscles following dissection and qRT-PCR performed using primers to detect Socs3 (B) IL-6 (C), TNF-a (D), CD68 (E), IFN-? (F) and F4/80 (G). (H) Representative CD68 (red) and DAPI (blue) immunostained sections of TA muscle from uninjured or day 1, 2, 3, 7, or 14 injured control and SOCS3 MscKO mice. (I) Representative F4/80 (green) and DAPI (blue) immunostained sections of TA muscle from uninjured or day 1, 2, 3, 7, or 14 injured control and SOCS3 MscKO mice. The proportion of CD68 (J) and F4/80 (K) positive nuclei were determined using Axiovision software. Data are expressed as mean ± SEM. Statistical analysis was performed using a two-way ANOVA with a Bonferonni’s post-hoc multiple comparisons test to determine the effects of genotype and time. n = 3–6 mice/time-point/genotype. *P < 0.05, **P < 0.01, ****P < 0.0001 compared to control.
Fig 2: Recognition of TAMs (tumor-associated macrophages) and M1 macrophages in tumor and inflammatory tissues by immunofluorescent imaging. A, F4/80(+) and CD206(+) indicated that TAMs were localized in tumor tissue. B, F4/80(+) and CD16(-) indicated that no M1 macrophages were detected in tumor tissue. C, Cyanine 7 deoxymannose (Cy7-DM) signals were overlapped with macrophages in tumor tissue, Cy7-DM(+) and F4/80(+). D, F4/80(+) and CD206(-) indicated that no TAMs were detected in inflammatory tissue. E, F4/80(+) and CD16(+) indicated that M1 macrophages were localized in inflammatory tissue. F, Merely Cy7-DM signal was found in inflammatory tissue with abundant macrophages.
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