c4 cedarlane cl7504f  (Cedarlane)

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Endothelial Cell–Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature

doi: 10.1161/ATVBAHA.118.311689

Figure Lengend Snippet: Complement deposition and histopathologic changes in retinas of aged Tspan12 ECKO mice. A , Complement factor C4 deposition around retinal blood vessels, inner nuclear layer (INL), and outer plexiform layer (OPL). White arrows point to some of the small cystoid lesions decorated with complement. B , H&E staining of a control retina and a relatively strongly affected area of a mutant retina reveals cystoid lesions consistent with edema. C , Increased F4/80 staining on microglia of mutant retinas. D through F , Extravastion of IgG, transferrin, and albumin in Tspan12 ECKO retinas. G , COL4 (collagen IV) staining in retinal blood vessels of 12-mo-old ECKO mice is increased, staining in the inner limiting membrane (white arrowheads) serves as internal staining control.

Article Snippet: The following primary antibodies and lectins were used for this study: GS Isolectin B4 Alexa-488 conjugate 1:100 (Invitrogen, I21411), rat PECAM (platelet endothelial cell adhesion molecule) 1:50 (BD, 550274), rat PLVAP 1:50 (BD, 550563), rabbit desmin 1:200 (Cell Signaling, D93F5), rat vascular endothelial (VE)-cadherin 1:100 (BD, clone 11D4), rat complement component C4 1:200 (Cedarlane, CL7504AP), goat transferrin 1:200 (R&D, AF3987), rabbit COL4 (collagen IV) 1:200 (Abcam, 6586), goat albumin 1:200 (Bethyl, A90-234A), goat IBA1 1:200 (Novus, NB100-1028), rat F4/80 1:200 (AbD Serotec, MCA497R), rabbit PKC (protein kinase C) α 1:200 (Santa Cruz, C-20), and rabbit Sox9 1:200 (Millipore, AB5535).

Techniques: Staining, Control, Mutagenesis, Membrane

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Endothelial Cell–Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature

doi: 10.1161/ATVBAHA.118.311689

Figure Lengend Snippet: Colocalization of complement factor C4 with several retinal cell types. A , Twelve-mo-old Tspan12 ECKO retinal cross-sections stained with anti-C4 and IB4. Split and merged channels are shown. Arrows indicate blood vessels decorated with C4. B , Colocalization of extravasated IgG and C4 on a subset of retinal cells and on the borders of cystoid lesions in the mutant tissue. C , Rod-bipolar cell spacing seems increased in the mutant tissue. The section shows also 1 or 2 C4-decorated Müller glia cells, which were recognized by their extensions towards the outer limiting membrane (arrow). D , Disorganized Müller cells in the mutant tissue. Sox9 marks nuclei of Müller glia. Arrow highlights a C4-decorated cell which was recognized as amacrine cell based on cell body location and cell shape. E , Partial colocalization of C4 with microglia marker IBA-1. PKC indicates protein kinase C; and Sox9, SRY-box 9.

Article Snippet: The following primary antibodies and lectins were used for this study: GS Isolectin B4 Alexa-488 conjugate 1:100 (Invitrogen, I21411), rat PECAM (platelet endothelial cell adhesion molecule) 1:50 (BD, 550274), rat PLVAP 1:50 (BD, 550563), rabbit desmin 1:200 (Cell Signaling, D93F5), rat vascular endothelial (VE)-cadherin 1:100 (BD, clone 11D4), rat complement component C4 1:200 (Cedarlane, CL7504AP), goat transferrin 1:200 (R&D, AF3987), rabbit COL4 (collagen IV) 1:200 (Abcam, 6586), goat albumin 1:200 (Bethyl, A90-234A), goat IBA1 1:200 (Novus, NB100-1028), rat F4/80 1:200 (AbD Serotec, MCA497R), rabbit PKC (protein kinase C) α 1:200 (Santa Cruz, C-20), and rabbit Sox9 1:200 (Millipore, AB5535).

Techniques: Staining, Mutagenesis, Membrane, Marker

Journal: Cell Death & Disease

Article Title: Clusterin facilitates apoptotic cell clearance and prevents apoptotic cell-induced autoimmune responses

doi: 10.1038/cddis.2016.113

Figure Lengend Snippet: Clu-deficient mice develop autoimmune symptoms in response to apoptotic cells. Aged-matched Clu −/− and WT mice were injected with apoptotic cells once a week for 5 weeks. Two weeks after the first injection of apoptotic cells, 4–6 mice per group were killed bimonthly. ( a ) Circulating IgG anti-dsDNA antibody levels were quantified. Results are expressed in kU/ml, mean±S.E.M., n =4 to 10; * P ≤0.05. Insert, IgG anti-dsDNA antibody titers were quantified by enzyme-linked immunosorbent assay in the serum of PBS-injected Clu −/− and WT mice. ( b ) Ten weeks after the first injection of apoptotic cells, kidney sections from Clu −/− and WT mice were stained with FITC-labeled anti-mouse IgG (left pictures) or unlabeled anti-C4 revealed with a FITC-labeled anti-rat IgG antibodies (right pictures). Glomeruli were stained with DAPI (circles). Results are representative of four mice. ( c ) Spleens of WT and Clu −/− mice were weighed 10 weeks after the first injection of apoptotic cells or PBS. Results are expressed in mg, mean±S.E.M., n =4; * P ≤0.05. ( d ) The expression of the mRNA encoding SAP was analyzed by quantitative PCR in the livers from Clu −/− and WT mice 10 weeks after the first injection of apoptotic cells or PBS. Results are expressed as a relative expression with GAPDH used as a calibrator, mean;±S.E.M., n =5; * P <0.05

Article Snippet: Tissue sections were stained with FITC-labeled goat anti-mouse IgG antibodies (Dako) or unlabeled rat anti-C4 mAb (Cedarlane, Burlington, Canada).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Expressing, Real-time Polymerase Chain Reaction