polyclonal cross-reactive anti-human cfh  (Quidel)

Journal: Molecular Immunology

Article Title: Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase

doi: 10.1016/j.molimm.2009.03.030

Figure Lengend Snippet: Renal C3 stain in C fh −/− mice 24 h after the injection of purified mCFH. Kidney sections were stained for C3 using polyclonal anti-mouse C3. Linear capillary wall staining is evident in C fh −/− mice injected with PBS or 0.75 μg of LPS. In contrast, mesangial staining is seen in C fh −/− mice 24 h after injection of 1 mg of mCFH. C3 staining is present along Bowman's capsule and within tubulo-interstitium of wild-type mice. Tubulo-interstitial staining is absent in C fh −/− mice injected with PBS or LPS. However, after administration of mCFH, tubulo-interstial staining is now evident in the C fh −/− animals. Original magnification 40×. WT: wild-type.

Article Snippet: After each chromatography step the samples were checked for the presence of CFH by western blot using polyclonal cross-reactive anti-human CFH (Quidel, CA, USA).

Techniques: Staining, Injection, Purification

Journal: Molecular Immunology

Article Title: Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase

doi: 10.1016/j.molimm.2009.03.030

Figure Lengend Snippet: Nature of C3 bound to the glomeruli 24 h after the administration of mCFH. (A) Kidney sections were stained for C3 using polyclonal anti-mouse C3d (red stain) or polyclonal anti-mouse C3 (green stain) antibodies. In the mice injected with PBS linear capillary wall staining was evident using both antibodies. However, in the C fh −/− mice injected with mCFH a mesangial staining pattern was evident using anti-mouse C3 antibody whilst the linear capillary wall staining pattern remained unchanged with the anti-C3d antibody. The merged images showed that the areas of mesangial reactivity did not co-stain with the anti-C3d. Original magnification 40×. (B) Western blot analysis of C3 under reducing conditions using solubilised laser dissected glomerular tissue from C fi −/− , C fh −/− or C fh −/− mice injected with mCFH. To demonstrate the positions of the intact C3 α -chain, the α ′ chain of C3b and the β -chain of C3 plasma from both wild-type (intact C3) and C fi −/− mice (in which all C3 is circulating as C3b) was also run on the gel. C3 α -chain fragments and the C3 β -chain were evident in C fh −/− mice with or without the administration of mCFH. In contrast no α -chain fragments were evident in glomeruli from C fi −/− animals. (C) Glomerular C3 staining in mice with combined deficiency of CFH and CFI (C fh −/− .C fi −/− ) that have been given CFI. Following adminstration of sera containing CFI, linear capillary wall C3 staining develops that is reactive with both the anti-C3 and anti-C3d antibody. Original magnification 40×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Article Snippet: After each chromatography step the samples were checked for the presence of CFH by western blot using polyclonal cross-reactive anti-human CFH (Quidel, CA, USA).

Techniques: Staining, Injection, Western Blot, Clinical Proteomics

Journal: Molecular Immunology

Article Title: Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase

doi: 10.1016/j.molimm.2009.03.030

Figure Lengend Snippet: Detection of CFH-related proteins in C fh −/− mice and tracking mCFH after administration to C fh −/− mice. (A) Serum western blot for CFH using cross-reactive polyclonal anti-human CFH antibody. (B) Renal sections from C fh −/− mice 2 h after the injection of Alexa-488-tagged CFH. At this time point the Alexa-488-tagged CFH was detected within the mesangium and tubulointerstitium but not along the GBM. At this time-point linear C3 staining is evident in C fh −/− mice similar to unmanipulated C fh −/− mice (far right panel). (C) Kidney sections immunostained for CFH using anti-human CFH antibody from wild-type, unmanipulated C fh −/− mice and C fh −/− mice 24 h after injection of mCFH. Some mesangial reactivity is present in wild-type glomeruli (left panel) whilst marked linear capillary wall staining is evident in the unmanipulated C fh −/− mice (middle panel). In contrast, a mesangial staining pattern was evident in C fh −/− mice 24 h after injection of mCFH (right panel) with very little staining along the capillary walls (arrows). Original magnification, 40×.

Article Snippet: After each chromatography step the samples were checked for the presence of CFH by western blot using polyclonal cross-reactive anti-human CFH (Quidel, CA, USA).

Techniques: Western Blot, Injection, Staining

Journal: Molecular Immunology

Article Title: Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase

doi: 10.1016/j.molimm.2009.03.030

Figure Lengend Snippet: Influx of neutrophils into the glomeruli after the administration of mCFH. (A) Glomerular neutrophil numbers in C fh −/− mice 24 h after injection of PBS, 0.75 μg of LPS or 1 mg of mCFH. Bars denote median values with standard deviation (B) Glomerular neutrophils evident in representative glomerular light microscopic images in mice that had received mCFH but not LPS. Original magnification 40× (C) FACS detection of blood neutrophils using rat IgG2b anti-mouse GR-1 antibody in Cfh −/− mice before, 24 and 48 h after neutrophil depletion. Neutrophil depletion was achieved by a single injection of rat monoclonal IgG2b anti-murine neutrophil Ly.6G (αNE). (D) Glomerular neutrophil numbers in neutrophil-depleted C fh −/− mice 24 h after injection of mCFH demonstrating absence of glomerular neutrophils in mice pre-treated with the αNE antibody. (E) Glomerular C3 stain using polyclonal anti-mouse C3 antibody in neutrophil-depleted mice treated with mCFH. The absence of neutrophils did not influence the change in glomerular C3 staining seen after the administration of mCFH. Compare this image with that shown in . Original magnification 40×. WT: wild-type.

Article Snippet: After each chromatography step the samples were checked for the presence of CFH by western blot using polyclonal cross-reactive anti-human CFH (Quidel, CA, USA).

Techniques: Injection, Standard Deviation, Staining