Mca928

The IgG₁ mouse anti-CR1 monoclonal antibody J3D3 was used to stain CR1 clusters, as it does not recognise epitopes in the LHR-D region^{44 (link)}, so was considered unlikely to influence any effect the Sl or McC polymorphisms might have on clustering. Two aliquots from each sample of washed, unfixed erythrocytes at 4% haematocrit in PBS/1% BSA were incubated with 5 μg/ml of either J3D3 or IgG₁ mouse anti-human isotype control (MCA928, AbD Serotec, Kidlington, UK) to give a final sample volume of 50 μl. Samples were incubated at 4 °C for one hour (resuspended every ten mins) and washed three times in 1 ml cold PBS (Oxoid, Basingstoke, UK). 50 μl of 4 μg/ml Alexa Fluor 488-conjugated goat anti-mouse secondary antibody was added and the sample was incubated at 4 °C in the dark for one hour (resuspended every ten mins) and subsequently washed three times in 1 ml cold PBS. The sample was resuspended at 30–40% haematocrit, smeared on a glass slide, air-dried, mounted under a 22 mm × 22 mm glass coverslip with 10 μl of DABCO-glycerol (2.5 mg DABCO (Sigma-Aldrich, Poole, UK), 0.5 ml glycerol and 0.5 ml PBS) and sealed with nail varnish. All steps to this point were undertaken at the KEMRI-Wellcome Trust Research Unit, Kilifi and the resultant slides were shipped to Edinburgh for confocal microscopy.

Primary antibodies tested targeted cell populations of astrocytes, microglia, neurons, T lymphocytes, B lymphocytes, and macrophages (Table 1). All primary antibodies were diluted in one of two commercial diluents (IHC Diluent (Novacastra, Leica); Dako Antibody Diluent (Dako, Glostrup, Denmark)). Two-fold serial dilutions of each antibody were tested to determine an optimal staining range. If the signal was weak to absent or background staining was present, additional dilution tests were performed until optimal staining was achieved. Antibodies were applied in a 37 °C, humidified incubator for 60–120 min or overnight (∼16 hrs) at 4 °C in a humidified, covered dish. After primary antibody incubation, slides were washed three times for 5 min each in 1 × PBS before secondary antibody application.
Negative controls for each primary antibody consisted of either an isotype-matched negative primary control (MCA928, AbD Serotec, Kidlington, UK) for monoclonal antibodies or rabbit serum for polyclonal antibodies.