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Fig. 1 | Journal of Neuroinflammation

Fig. 1

From: Complement C3a activates astrocytes to promote medulloblastoma progression through TNF-α

Fig. 1

Complement C3a is present in MB tissue. Human and mouse MB sections underwent IHC staining for C3 and C3a. For detecting C3, anti-human/mouse C3 polyclonal antibody (GeneTex) was used, and for detecting C3a, the neoepitope recognizing antibodies were used as follows: anti-human C3a monoclonal antibody, clone 2991 (Bio-Rad) and anti-mouse C3a monoclonal antibody, clone I87-1162 (BD Biosciences). a Representative IHC staining of intact C3 (left) and C3a (right) in human Hh-type MB tissue; brown color displays the positive staining. N normal adjacent tissue; T tumor area. The yellow line defines the boundary between normal and tumor area. b Left upper and middle panels show the whole mount of WT normal brain and tumor-bearing brain of primary MB mouse (pMB), respectively, with normal cerebellum or MB tumor framed. The left lower panel shows the dissected tumor mass from the subcutaneously transplanted MB model (Subq). The 2nd panels show H&E staining of paraffin-embedded sections of the above murine MB tissue. Frames line out the area for subsequent IHC staining images. The 3rd and 4th panels show representative IHC staining of intact C3 and C3a in murine MB tumor tissue. c Quantitative PCR was performed to determine C3 mRNA levels in the normal cerebellum of WT mice and in primary MB tissue. d C3a protein levels in the WT normal cerebellum, primary MB tissue and subcutaneously transplanted MB tissue were assessed by ELISA. ***p < 0.001 vs. WT group in c and d. Conventional RT–PCR (e) and quantitative RT–PCR (f) were performed to detect C3a receptor (C3aR, gene: C3AR1) expression in primary astrocytes (pAS), tumor-associated astrocytes (TAAs), purified MB cells (MB) and mouse primary microglia (Mg); GAPDH served as an internal control. ***p < 0.001 vs. MB cells group and ### p < 0.001 vs. pAS group in f. g Frozen sections were prepared with primary murine MB tumor tissues, and immunostaining of C3aR (in red) and GFAP (in green) or Iba-1(in green) were performed to determine the C3aR distribution. Values are expressed as the mean ± SEM from at least three independent experiments in c, d and f. (IHC: immunohistochemistry)

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