Fig 1: Effect of neutralizing antibodies for chemokines/chemoattractans in monocytes/MFs in vitro cell migration assays. (A) The effect of the neutralizing antibodies against CCL2 (500 ng/mL), CCL3 (500 ng/mL), and CCL21 (500 ng/mL) in the monocytes/MFs migration assay under the chemoattractant effect of the CM of the glomeruli from DN rats. HAM-F10 and 5% FBS were used as negative and positive control, respectively, of in vitro stimuli for migration. DAPI was used to stain the nuclei of monocytes/MFs. Representative quadrants show monocytes/MFs in blue (400× magnification). (B) Quantification of positive intraglomerular area (%) of deconvoluted color for DAB in (A). Graphs represent distribution of each sample and the mean ± S.D. * p < 0.05, and *** p < 0.001 versus Ctrl rats. # p < 0.05 and ### p < 0.001, DN + MRS1754 versus DN rats. n = 4.
Fig 2: Expression of chemokines/chemoattractans in glomeruli of DN + MRS1754 rats. (A) Protein expression of CCL3 and CCL21 (green color) by immunohistofluorescence in glomeruli (red color) of Ctrl, DN, and DN + MRS1754 rats. The dotted lines represent glomeruli areas. (B) Quantification of positive intraglomerular area (%) of (A). Graphs represent the mean ± S.D. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus Ctrl rats. # p < 0.05 and ## p < 0.01, DN + MRS1754 versus DN rats. n = 4.
Fig 3: Chemokines/chemoattractans secreted by glomeruli of DN + MRS1754 rats. Quantification of (A) CCL2, (B) CCL3, (C) CCL6, (D) CCL21, and (E) CXCL9 by ELISA in the CM of glomeruli isolated from Ctrl, DN, and DN + MRS1754 rats. Measures of each chemokine/chemoattractant were normalized by H3 quantification by western blot (Figure S1). Graphs represent distribution of each sample and mean ± S.D. * p < 0.05, and ** p < 0.01 versus Ctrl rats. # p < 0.05, ## p < 0.01, ### p < 0.001 DN + MRS1754 versus DN rats. n = 6 samples per duplicate.
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