Goat anti mouse igg1

The staining of Col I (anti-collagen I antibody, 1:2000, Abcam, Cambridge, UK) and αSMA (anti-actin α-smooth muscle, 1:400, Sigma) was evaluated after 7 and 14 d in SC monocultures on scPLCL and scPLCL_A2P. Additionally, in cocultures of ECs and SCs on scPLCL_A2P, the staining of pancytokeratin (AE1/AE3, 1:250, Cytokeratin Pan Ab, Thermo Fisher Scientific) and actin cytoskeleton organization (phalloidin-tetramethylrhodamine B isothiocyanate, 1:500, Sigma-Aldrich) was evaluated after 7 and 14 d of cell culturing.
The samples were fixed with 0.2% Triton X-100 (Sigma-Aldrich) in 4% PFA (Sigma-Aldrich) and incubated overnight in the abovementioned primary antibody dilutions. The following day, the SC monocultures were incubated in secondary antibody dilutions (1:400, goat anti-mouse IgG1 or 1:300, goat anti-mouse IgG (H + L), Alexa-fluor 488, green fluorescence, Invitrogen). The EC and SC cocultures were incubated in a mixture of secondary antibody (1:400, goat anti-mouse IgG (H + L), Alexa-fluor 488, green fluorescence, Invitrogen) and phalloidin. Finally, the cell nuclei were stained with DAPI (1:200, blue fluorescence, Sigma-Aldrich), and the samples were imaged with a fluorescence microscope (Olympus).

Enucleated eyes were prepared for immunohistochemistry by embedding eyes in optimal cutting temperature medium (VWR, UK) and rapid freezing with isopentane on dry ice following trans-cardial perfusion. Eyes were stored at −20 °C until use. Immunohistochemistry experiments were carried out in 20 μm cryo-sections of fresh-frozen eyes as described previously^{14 (link)}. Sections were stained with primary antibodies against rat anti-mouse FcγRI (Clone 152-9, a kind gift from Dr Alison Tutt, University of Southampton), rabbit anti-human GFAP (Dako, Denmark), laminin (L9393, Sigma, UK) and VEGF (MA5-12184, Invitrogen). Secondary antibodies used were donkey anti-rat IgG-AF488, goat anti-rabbit IgG-AF568 (ThermoFisher, UK), goat anti-mouse IgG1 AF488, goat anti-rabbit IgG-AF568 (A11011, Invitrogen) and 4′6′-diamino-2-phenylindole (DAPI) for nuclei staining. Z-stack images acquired using a Leica SP8 (Leica Microsystems, IL, USA) confocal laser scanning microscope were converted into a maximum intensity projection in ImageJ (NIH, USA). Fluorescence was quantified in the whole field of view for separate channels using the histogram analysis function in ImageJ (NIH, USA) and shown as mean, modal, minimum and maximum values.

Seven-micrometer-thick cryosections were cut from the OCT-embedded biopsy specimen using a cryostat (Leica CM1950). The cryosections were then mounted onto microscope glass slides (VWR International), air-dried for 1 h, and stored at −80°C. Pre and post samples were placed on the same glass slide to minimize variations in staining efficiency. To stain the muscle capillaries, the slides were fixed in 4% paraformaldehyde (PFA), washed in phosphate buffered saline (PBS) and thereafter incubated for 2 h with primary antibodies against laminin (1:50; D18, DSHB, United States) and CD31/PECAM (1:400; JC70, Santa Cruz Biotechnology, United States) diluted in PBS containing 5% normal goat serum (NGS) and 0.02% Triton X-100. After being washed in PBS, secondary antibodies (1:500; 350 goat anti-mouse IgG2A and 1:1,000; 594 goat anti-mouse IgG1, Alexa Fluor, Invitrogen, United States) diluted in PBS containing 1% NGS were applied. The slides were mounted with a cover slip and Prolong Gold Antifade Reagent (Invitrogen, United States). This protocol stained the cell borders in blue and the muscle capillaries in red, as depicted in Figure 1.

Immunocytochemical examination was performed as previously described [37 (link)]. Cell cultures were fixed with 4% paraformaldehyde (PFA; USB Products, OH, USA) for 30 min and washed with phosphate-buffered saline (PBS). Fixed cells were blocked with 5% normal goat serum (Millipore, CA, USA) and 0.2% Triton X-100 (Amresco, OH, USA) in PBS for 30 min and incubated with antibodies against βIII Tubulin (TuJ1, mouse monoclonal antibody, 1:1000; Sigma-Aldrich), glial fibrillary acidic protein (GFAP, rabbit polyclonal antibody, 1:1000; Dako, Copenhagen, Denmark), S100β (mouse monoclonal antibody, 1:500; Sigma-Aldrich), and Ki67 (rabbit monoclonal antibody, 1:400; Thermo Scientific, CA, USA) for 1 h. After PBS rinses, the cells were incubated for 30 min with secondary antibodies conjugated to Alexa Fluor 488 (goat anti-mouse immunoglobulin G [IgG], 1:1000; Invitrogen), Alexa Fluor 546 (goat anti-mouse IgG₁, 1:1000; Invitrogen) or Cy3 (goat anti-rabbit IgG, 1:1000; Jackson ImmunoResearch, PA, USA) followed by 5 min in 4′,6-diamidino-2-phenylindole (DAPI, 1:10000 in PBS; Sigma-Aldrich) to stain the nuclei. Images were obtained using an inverse fluorescence microscope (DMIL; Leica, Hesse, Germany). TuJ1-, GFAP-, S100β-, or Ki67-positive cells were counted and normalized to total DAPI-positive cell numbers.

First, mouse IgG1 was isolated from the sera of highly IgG1-ANA-positive aged lupus-prone MRL-lpr/lpr mice (n = 20; age ≥ 15 weeks) (lupus IgG1) or age-matched control C57BL/6J mice (n = 20) (control IgG1) by immunoaffinity column chromatography using goat anti-mouse IgG1 (Thermo Fisher Scientific, Inc., San Jose, CA, USA) coupled to CNBr-activated Sepharose™ 4B (GE Healthcare, Chicago, USA) and quantified using the Pierce™ BCA Protein Assay Kit. The homogeneity and reactivity of the IgG1 antibody mixtures were analyzed by SDS-PAGE and IF staining with HEp-2 cells.
Second, the experimental design and time lines were followed as shown in Figure 4A. Briefly, eighteen 10-week-old female MRL-lpr/lpr mice were divided equally into three groups which were defined as the non-treated group, control IgG1 group (treated with IgG1 from age-matched control mice), and lupus IgG1 group (treated with IgG1 from aged MRL-lpr/lpr mice). Purified IgG1 was administered intravenously via the tail vein at 1.0 mg/(mouse∙week) for 10–18 weeks of age. Samples (blood, urine, spleen, and kidney) were collected at the indicated time points. All mice were sacrificed at 19 weeks of age for sample collection.