Cappel

Seven rats in total were used for immunohistochemical study. One week after PBS or TNF injection, optic nerve samples from the NG (3 rats) and HG (4 rats) groups were fixed by immersion in 10% neutral-buffered formalin for 24 h, dehydrated, embedded in paraffin, and sectioned (4 μm thick). Deparaffinized sections were incubated with 1% bovine serum and then reacted with primary antibodies against LC3 (1:200; Medical & Biological Laboratories Co.), or neurofilament-L (a marker of neurons; 1:100; DAKO Corporation, Carpinteria, CA, USA) diluted in 1% bovine serum overnight at 4°C. Sections were then exposed to the following secondary antibodies: FITC-labeled anti-rabbit antibody (1:100; Cappel, MP Biomedicals, LLC) or rhodamine-labeled anti-mouse antibody (1:100; Cappel, MP Biomedicals, LLC). The samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vesctashield with DAPI, Vector Laboratories, Inc., Burlingame, CA, USA). Negative controls were performed by replacing the primary antibody with PBS or serum.

IE were opsonised at a concentration of 5 x 10⁷ trophozoites/mL for 30 minutes at room temperature with rabbit anti-human erythrocyte antibodies (Cappel, MP Biomedicals, LLC; Santa Anna, CA, USA) using a sub-agglutinating 1/800 dilution of antibody in phosphate-buffered saline (PBS). In some experiments CS2 IE were opsonised with 20 % human immune serum from a pool of sera prepared from pregnant woman with placental malaria enrolled in the VT cohort in Papua New Guinea [31 (link)]. Opsonised cells were washed in fluorescence-activated cell sorting (FACS) wash buffer (PBS, 2 % new born calf serum) and resuspended in PBS (2 x 10⁸/mL) then stained with 10 μg/mL ethidium bromide (EtBr) for 30 minutes at room temperature. Following labeling with EtBr, cells were washed three times with cold FACS wash buffer and used immediately.

Binding of soluble FcαRI or IgA Ab to plate‐bound peptides was tested with Pepscan‐based ELISA's, which were adapted according to the method previously described 19, 43, 44. The samples were washed to remove unbound fragments after each incubation step. The 455‐well credit‐card format polypropylene cards containing the covalently linked IgA‐peptides were incubated with blocking solution (4% horse serum, 5% ovalbumin (w/v) in PBS/1% Tween). Next, peptides were incubated with soluble FcαRI or 293T cells transfected with FcαRI and subsequently with mouse anti‐human FcαRI IgG mAb (1 μg/ml; BD, Franklin Lakes, NJ). Then, peptides were incubated with rabbit anti‐mouse IgG‐HRP (1/1000, Dako, P0212) for 1 h at RT. Alternatively, after blocking, the covalently linked FcαRI‐peptides were incubated with pooled human serum IgA (Cappel™, MP Biomedicals, Santa Ana, CA), and rabbit anti‐human IgA‐HRP (1 μg/mL, Dako, P0212) was added. The peroxidase substrate 2,2′‐azino‐di‐3‐ethylbenzthiazoline sulfonate (ABTS) and 2 μL of 3% H₂O₂ was added (1 h, RT). Colour development was measured, which was quantified with a charge coupled device (CCD)‐camera and an image processing system. Values mostly ranged from 0 to 3000, a log scale similar to 1–3 of a standard 96‐well plate ELISA‐reader.