Table 1.
Details of primary and secondary antibodies used.
Fig 1.
Changes in EAE-induced cellular infiltration in the optic nerve.
A-D) Representative images of H&E stained sections from control and EAE optic nerves. Increased infiltration was evident in WT EAE sections, while A2 deletion markedly reduced this effect. E-H) High magnification images of boxed regions demonstrate EAE-induced cellular infiltration. Arrows indicate clusters of infiltrated cells. I) Quantification of infiltrated cells using ImageJ. Data are presented as Mean ± SEM. #p<0.01 WT EAE vs WT Con and *p<0.05 A2-/- EAE vs WT EAE. Number of animals used: 8(WT Control);12 (WT EAE); 9 (A2-/- EAE); 7 (A2-/- Control). Representative images are presented. Scale bar 50 μm.
Fig 2.
EAE-induced microglial/macrophage activation is ameliorated in the optic nerve of A2 knockout mice.
A-D) Immunofluorescence images of Iba1 labeled optic nerve sections showing increases in cells with activated morphology in response to EAE induction. The deletion of A2 significantly reduced this effect. E-H) Magnified images of the boxed regions demonstrate changes in the morphology of Iba1 positive cells and the effects of A2 deletion. Asterisks indicate Iba1 positive cells with activated morphology following EAE induction. I) Quantification of Iba1 positive cells with activated morphology. Data are presented as Mean ± SEM. #p<0.01 WT EAE vs WT Con and # p<0.01 A2-/- EAE vs WT EAE. Number of animals used: 8(WT Control);12 (WT EAE); 9 (A2-/- EAE); 7 (A2-/- Control). Representative images are presented. Scale bar 50 μm.
Fig 3.
A2 deletion suppressed EAE-induced upregulation of F4/80 cells.
A-D) Representative confocal images of optic nerve sections immunostained using F4/80 antibody. WT EAE optic nerves showed an increase in macrophage/monocyte, while A2 deletion greatly suppressed this effect. E-H) Magnified images of boxed areas represent increase in the presence of F4/80 positive cells induced by EAE. I) Quantification of F4/80 positive cells demonstrate the impact of A2 deletion on the number of macrophages induced by EAE. Optic nerve sections stained using antibodies against CD 16/32 (J-M), and CD 86 (O-R) and quantification (N&Q) showed upregulation of M1 macrophages in WT EAE samples which was significantly reduced by the A2 deletion. Studies using an M2 marker, A1, did not show any significant changes across the groups (T-W) Number of animals used in the study: Number of animals used: 8(WT Control);12 (WT EAE); 9 (A2-/- EAE); 7 (A2-/- Control). Representative images are presented. Scale bar 50 μm. Data are presented as Mean ± SEM. #p<0.01 WT EAE vs WT Con and # p<0.01 A2-/- EAE vs WT EAE.
Fig 4.
Deletion of A2 reduced EAE-induced axonal degeneration.
A-D) Confocal images of optic nerve sections from WT EAE mice demonstrate disorganized and damaged axons in the EAE optic nerves. Asterisks (*) indicate areas of axonal damage. E) Quantification of SMI 32 fluorescence intensity using ImageJ. Number of animals used: 8(WT Control);12 (WT EAE); 9 (A2-/- EAE); 7 (A2-/- Control). Representative images are presented. Data are presented as mean ± SEM. #p<0.01 WT EAE vs WT Con and A2-/- EAE vs WT EAE is non-significant. Scale bar 20 μm.
Fig 5.
A2 deletion protected against EAE-induced demyelination.
A-D) Representative images showing MBP staining. E-H) Higher magnification images from boxed areas showing the loss of myelin in WT EAE optic nerves while A2 deletion reduced the EAE-induced-demyelination. I) Quantification of immunofluorescence intensity of MBP. Number of animals used: 8(WT Control);12 (WT EAE); 9 (A2-/- EAE); 7 (A2-/- Control). Representative images are presented. Data are presented as mean ± SEM. #p<0.01 WT EAE vs WT Con and *p<0.05 A2-/- EAE vs WT EAE. Scale bar 50 μm.
Fig 6.
EAE-induced astrogliosis is reduced in A2 deficient optic nerves.
A-D) Representative confocal images optic nerve sections demonstrate the activation of astrocytes (labeled by GFAP antibody) in the WT EAE optic nerves while the deletion of A2 significantly suppressed the extent of astrogliosis. E-H) Magnified images of respective boxed areas in A-D demonstrate hypertrophic astrocytes. I) Quantification of glial activation in optic nerve sections of different groups. Scale bar 50 μm. Number of animals used: 8(WT Control); 12 (WT EAE); 9 (A2-/- EAE); 7 (A2-/- Control). Representative images are presented. Data are presented as mean ± SEM. #p<0.01 WT EAE vs WT Con and *p<0.05 A2-/- EAE vs WT EAE.