Journal: The Journal of Neuroscience

Article Title: Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve

doi: 10.1523/jneurosci.0555-21.2021

Figure Lengend Snippet: Figure 5. Marked increase in CR3 near the site of ONI. Complement C3b phagocytosis receptor CR3 is microglia/monocyte-specific, and CR31 microglia/monocyte number and phagocytic activation increase within the injured optic nerve. A, Representative images of CR3 immunolabeled uninjured (–) and injured (1, 3, 5, 7, 14 DPI) optic nerves at the injury site. Indicated regions (☐) are shown enlarged to the right of each image. Note the accumulation of CR31 cells and the increased cellular signal intensity following ONI. B, C, Quantitation confirms that the approximate number of CR31 cells (B) increases rapidly (by 1 DPI) and is sustained for at least 14 DPI, with a 30-fold peak at 5 DPI; likewise, CR3 immunofluorescence intensity (C) is elevated at all postinjury time points evaluated (1-14 DPI). D, The specificity of the CR3 (CD11b) antibody was verified using 14 DPI nerves from CR31/1 (left) and CR3/ mice (right). E, The approximate number of Iba11 microglia/monocytes in the nerve increases dramatically by 5 DPI and remains elevated until at least 14 DPI. F, Quantitation of CR31Iba1 (red), CR3-Iba11 (green), and colocalized CR31Iba11 (yellow) cells at/near the injury site. While most CR31 optic nerve cells are microglia, some CR31 cells in the injured nerve are likely infiltrating peripheral blood monocytes. G, CR3 (red) colocalization with Iba1 (green; microglia) at nerve injury site (3, 5 DPI), in the distal nerve (3 DPI), and in the chiasm (14 DPI). H–J, Presence of myeloid cell markers CD206 (H), CD45 (I), and P2RY12 (J) in 14 DPI optic nerves. Iba11 and CR31 microglia/monocytes at/near the injury are large, round, and lack processes: morphologic changes associated with phagocytic activation. L, Similarly, these Iba11 (green) microglia/monocytes express strikingly high levels of lysosome marker CD68 (red; observed 3-14 DPI, 14 DPI shown). M, C1q deletion dampens the CR31 cell response to injury plus zymosan 1 CPT-cAMP treatment at 5 and 14 DPI. N, Pro-regenerative treatments may augment the CR31 cell response in the injured nerve at 14 DPI. Scale bars: A, 50 mm, 10 mm; D, G, H, I, J, L, 50 mm. Graphs represent either mean 6 SEM or 25th to 75th percentile (box), median (line), range (whiskers), and individual data points (circles). B, C, E, K, *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, one-way ANOVA with Dunnett post-test vs uninjured or untreated. J, **p , 0.01, t test.

Article Snippet: Antibodies used in immunohistochemistry experiments Antibody Target (specificity) Host species Manufacturer Dilution C1q Complement C1q (C1q) Rabbit Abcam #ab182451 1:250 C3 Complement C3 (C3) Goat MP/Cappel #55730 1:1000 C3d Complement C3d (C3d opsonin) Rabbit Dako #A0063 1:1000 CC1 CC1, APC (mature oligodendrocytes) Mouse Abcam #ab16794 1:100 CD11b Complement receptor 3, CR3, Mac1, integrin aMb 2 (monocytes, microglia, PMNs) Rat Bio-Rad #MCA711G 1:1000 CD45 PTPRC (microglia, monocytes, PMNs, NKs, lymphocytes) Rat Novus Biologicals #NB100-77417 1:500 CD68 Lysosomes (phagocytes) Rat Bio-Rad #MCA1957 1:200 CD206 Mannose receptor (monocytes, microglia) Goat R&D Systems #RB01 1:200 GAP43 Growth-associated protein 43 (regenerating axons) Sheep Custom Variable GFAP Glial fibrillary acidic protein (astrocytes) Rabbit Dako #Z0334 1:5000 GFAP Glial fibrillary acidic protein (astrocytes) Mouse Sigma #G3893 1:2500 Iba1 Ionized calcium binding adaptor molecule 1 (microglia, monocytes) Rabbit Wako #01919741 1:500 MBP Myelin basic protein (myelin) Rabbit Abcam #ab40390 1:500 Olig2 Oligodendrocyte transcription factor 2 (oligodendrocytes) Goat R&D Systems #AF2418 1:500 P2Y12 Purinergic receptor P2Y (microglia) Rabbit AnaSpec #AS-55043A 1:500 TUJ1 b -III tubulin (RGCs) Rabbit Abcam #ab18207 1:500 mean RGC count for each retina was transformed to RGC survival density (RGCs/mm2, divided by 0.235 mm2 image area), then expressed as %WT control.

Techniques: Activation Assay, Immunolabeling, Quantitation Assay, Immunofluorescence, Marker

Journal: The Journal of Neuroscience

Article Title: Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve

doi: 10.1523/jneurosci.0555-21.2021

Figure Lengend Snippet: Figure 6. Comparatively modest CR31 cell response in the retina after ONI. Although CR31 microglia and phagocytic activation both increase after ONI, the retinal cellular inflammatory response is less robust than that in the nerve. A, B, Images of retinal whole mounts (A) and retinal cross sections (B) from uninjured (–) and ONI (1, 3, 5, 7 DPI) mice, immunolabeled for Iba1 (microglia, green) and CD68 (lysosomes, red), showing increased microglial number and activation after ONI. C, Quantitation of Iba11 microglia in the indicated laminae over time indicates that microglia accumulate in the IPL and GCL, peaking at 5 DPI. D, After ONI, Iba11 microglia within the GCL 1 IPL demonstrate changes in morphology associated with phagocytic activation. E, Elevated CD681 lysosomal content. Images (F) and quantitative analysis (G) of complement C3b phagocytosis receptor CR3 (red) near exclusive colocalization (yellow) with Iba11 (green) cells in retina. In both the uninjured and injured retinas, all CR31 cells appear to be microglia. H, CR31 microglia accumulate within the GCL 1 IPL after ONI. I, J, Representative images of Iba11 (I) and CR31 (J) microglia/monocytes in optic nerve head after ONI (nerve, retina, and retinal laminae marked). Note stronger microglia/monocyte response in the optic nerve head versus the retina. K, Retinal and optic nerve sections from the same 5 DPI mouse, proc- essed in parallel, reveal a comparatively stronger Iba11 (green) and CR31 (red) cell responses in the nerve. Indicated regions (☐; purple represents retina; orange represents optic nerve head; yellow rep- resents nerve) are enlarged below. Compared with the ONI site, the microglia/monocyte inflammatory response in the distal nerve and optic nerve head is moderate, and in the retina it is weak. Scale bars: A, B, F, 10 mm; I, J, 50 mm; K, 300 mm, 20 mm. Graphs represent either mean 6 SEM or 25th to 75th percentile (box), median (line), range (whiskers), and individual data points (circles). *p, 0.05, **p, 0.01, ****p, 0.0001, one-way ANOVA with Dunnett post-test vs uninjured or untreated.

Article Snippet: Antibodies used in immunohistochemistry experiments Antibody Target (specificity) Host species Manufacturer Dilution C1q Complement C1q (C1q) Rabbit Abcam #ab182451 1:250 C3 Complement C3 (C3) Goat MP/Cappel #55730 1:1000 C3d Complement C3d (C3d opsonin) Rabbit Dako #A0063 1:1000 CC1 CC1, APC (mature oligodendrocytes) Mouse Abcam #ab16794 1:100 CD11b Complement receptor 3, CR3, Mac1, integrin aMb 2 (monocytes, microglia, PMNs) Rat Bio-Rad #MCA711G 1:1000 CD45 PTPRC (microglia, monocytes, PMNs, NKs, lymphocytes) Rat Novus Biologicals #NB100-77417 1:500 CD68 Lysosomes (phagocytes) Rat Bio-Rad #MCA1957 1:200 CD206 Mannose receptor (monocytes, microglia) Goat R&D Systems #RB01 1:200 GAP43 Growth-associated protein 43 (regenerating axons) Sheep Custom Variable GFAP Glial fibrillary acidic protein (astrocytes) Rabbit Dako #Z0334 1:5000 GFAP Glial fibrillary acidic protein (astrocytes) Mouse Sigma #G3893 1:2500 Iba1 Ionized calcium binding adaptor molecule 1 (microglia, monocytes) Rabbit Wako #01919741 1:500 MBP Myelin basic protein (myelin) Rabbit Abcam #ab40390 1:500 Olig2 Oligodendrocyte transcription factor 2 (oligodendrocytes) Goat R&D Systems #AF2418 1:500 P2Y12 Purinergic receptor P2Y (microglia) Rabbit AnaSpec #AS-55043A 1:500 TUJ1 b -III tubulin (RGCs) Rabbit Abcam #ab18207 1:500 mean RGC count for each retina was transformed to RGC survival density (RGCs/mm2, divided by 0.235 mm2 image area), then expressed as %WT control.

Techniques: Activation Assay, Immunolabeling, Quantitation Assay

Journal: The Journal of Neuroscience

Article Title: Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve

doi: 10.1523/jneurosci.0555-21.2021

Figure Lengend Snippet: Figure 9. Complement is produced by microglia and other cells in the injured optic nerve. A, Representative images of 1 and 7 DPI nerve sections after RNAscope using a C1q mRNA probe. A small population of local cells produces C1q shortly after ONI, as do many of the cells that subsequently accumulate within the injury site. B, Representative images of 7 DPI adjacent sections from the same nerve processed for C1q mRNA (top) and CR3 protein (bottom) suggest C1q production by CR31 microglia/monocytes within the injury site. C, Representative image of 7 DPI nerve section after RNAscope procedure using C1q (red) and C3 (green) mRNA probes followed by immunohistochemistry using CD68 (white) antibody. Most C1q1 cells and many C31 cells are also CD681, suggesting that phagocytic monocytes in and near the injury site contribute to ONI-induced complement elevation. D, Sham-injury (no ONI) and 14 DPI nerve sections from mice treated with control (left) or PLX5622 (right) chow, immunostained for Iba1 and CR3. PLX nearly eliminates microglia in the sham-injury (no ONI) optic nerve, but is not as effective after ONI, especially for CR31 microglia/monocytes at/near the injury site. E–G, ONI and PLX treatment each affects local expression of Aif1 (Iba1 microglia/monocytes, E), C1qa (C1q, F), and C3 (G) within the nerve, as analyzed by qPCR. The reduction in C1q (but not C3) mRNA with PLX implicates microglia in local naive and injury-associated C1q production. H, Sham-injury (no ONI) and 14 DPI retinal whole mounts from mice treated with control (left) or PLX (right) food. PLX nearly eliminates microglia in both sham and ONI retinae. I, J, qPCR analysis reveals that ONI and PLX treatment each affects expression of Aif1 (Iba11 microglia/monocytes, I) and C1qa (C1q, J) within the retina, and points to microglia as the primary source of retinal C1q expression. Scale bars: A–D, H, 50 mm. N values: 4/group for retina qPCR, 2 or 3/group for nerve qPCR. Data are mean 6 SEM. Two-way ANOVA (main effects of injury time and PLX treatment) with Dunnett or Sidak post-test: *p , 0.05, ****p , 0.0001 vs same chow sham-injured; ##p , 0.01, ###p , 0.001, ####p , 0.0001 vs control chow at same time point.

Article Snippet: Antibodies used in immunohistochemistry experiments Antibody Target (specificity) Host species Manufacturer Dilution C1q Complement C1q (C1q) Rabbit Abcam #ab182451 1:250 C3 Complement C3 (C3) Goat MP/Cappel #55730 1:1000 C3d Complement C3d (C3d opsonin) Rabbit Dako #A0063 1:1000 CC1 CC1, APC (mature oligodendrocytes) Mouse Abcam #ab16794 1:100 CD11b Complement receptor 3, CR3, Mac1, integrin aMb 2 (monocytes, microglia, PMNs) Rat Bio-Rad #MCA711G 1:1000 CD45 PTPRC (microglia, monocytes, PMNs, NKs, lymphocytes) Rat Novus Biologicals #NB100-77417 1:500 CD68 Lysosomes (phagocytes) Rat Bio-Rad #MCA1957 1:200 CD206 Mannose receptor (monocytes, microglia) Goat R&D Systems #RB01 1:200 GAP43 Growth-associated protein 43 (regenerating axons) Sheep Custom Variable GFAP Glial fibrillary acidic protein (astrocytes) Rabbit Dako #Z0334 1:5000 GFAP Glial fibrillary acidic protein (astrocytes) Mouse Sigma #G3893 1:2500 Iba1 Ionized calcium binding adaptor molecule 1 (microglia, monocytes) Rabbit Wako #01919741 1:500 MBP Myelin basic protein (myelin) Rabbit Abcam #ab40390 1:500 Olig2 Oligodendrocyte transcription factor 2 (oligodendrocytes) Goat R&D Systems #AF2418 1:500 P2Y12 Purinergic receptor P2Y (microglia) Rabbit AnaSpec #AS-55043A 1:500 TUJ1 b -III tubulin (RGCs) Rabbit Abcam #ab18207 1:500 mean RGC count for each retina was transformed to RGC survival density (RGCs/mm2, divided by 0.235 mm2 image area), then expressed as %WT control.

Techniques: Produced, RNAscope, Immunohistochemistry, Control, Expressing

Journal: The Journal of Neuroscience

Article Title: Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve

doi: 10.1523/jneurosci.0555-21.2021

Figure Lengend Snippet: Figure 10. Complement proteins associate with multiple cell types after ONI. A–F, Images of immunolabeled optic nerves showing association of complement proteins with microglia/mono- cytes (A, red C1q, green CR3; B, green C3, red CR3), astrocytes (C, red C1q, green GFAP), endothelial cells (D, red C1q, green IB4), and oligodendrocytes (E, red C1q, green Olig2; F, red C1q, green CC1) in the naive and injured optic nerve, at and beyond the injury site. Indicated region (☐) is enlarged below. Arrowheads indicate colocalization (yellow). Double arrows indicate nearby localization. G–M, Retinal cross-sections showing association of immunolabeled complement proteins with microglia/monocytes (G, red C1q, green CR3; H, green C3, red CR3; I, red C1q, green CD68), RGCs (J, red C1q, green TUJ1; K, green C3, red TUJ1), astrocytes (L, red C1q, green GFAP), and endothelial cells (M, red C1q, green IB4) in the retina after ONI. Arrowheads indicate colocalization (yellow). Scale bars: A, B, D–M, 10 mm; C, 50 mm.

Article Snippet: Antibodies used in immunohistochemistry experiments Antibody Target (specificity) Host species Manufacturer Dilution C1q Complement C1q (C1q) Rabbit Abcam #ab182451 1:250 C3 Complement C3 (C3) Goat MP/Cappel #55730 1:1000 C3d Complement C3d (C3d opsonin) Rabbit Dako #A0063 1:1000 CC1 CC1, APC (mature oligodendrocytes) Mouse Abcam #ab16794 1:100 CD11b Complement receptor 3, CR3, Mac1, integrin aMb 2 (monocytes, microglia, PMNs) Rat Bio-Rad #MCA711G 1:1000 CD45 PTPRC (microglia, monocytes, PMNs, NKs, lymphocytes) Rat Novus Biologicals #NB100-77417 1:500 CD68 Lysosomes (phagocytes) Rat Bio-Rad #MCA1957 1:200 CD206 Mannose receptor (monocytes, microglia) Goat R&D Systems #RB01 1:200 GAP43 Growth-associated protein 43 (regenerating axons) Sheep Custom Variable GFAP Glial fibrillary acidic protein (astrocytes) Rabbit Dako #Z0334 1:5000 GFAP Glial fibrillary acidic protein (astrocytes) Mouse Sigma #G3893 1:2500 Iba1 Ionized calcium binding adaptor molecule 1 (microglia, monocytes) Rabbit Wako #01919741 1:500 MBP Myelin basic protein (myelin) Rabbit Abcam #ab40390 1:500 Olig2 Oligodendrocyte transcription factor 2 (oligodendrocytes) Goat R&D Systems #AF2418 1:500 P2Y12 Purinergic receptor P2Y (microglia) Rabbit AnaSpec #AS-55043A 1:500 TUJ1 b -III tubulin (RGCs) Rabbit Abcam #ab18207 1:500 mean RGC count for each retina was transformed to RGC survival density (RGCs/mm2, divided by 0.235 mm2 image area), then expressed as %WT control.

Techniques: Immunolabeling

Journal: The Journal of Neuroscience

Article Title: Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve

doi: 10.1523/jneurosci.0555-21.2021

Figure Lengend Snippet: Figure 11. CR31 phagocytic microglia/monocytes alter the local environment of the injured nerve. A, Representative images of optic nerves (1, 5, 7 DPI) immunolabeled for CR3 (second row, red in merged) and MBP (third row, green in merged). B, Single confocal planes with orthogonal views of MBP1 myelin debris inside CR31 cells (14 DPI). C, Representative images of injured optic nerves (1, 5 DPI) immunolabeled for CD68 (second row, red in merged) and MBP (third row, green in merged), which strongly resemble the CR3 and MBP pattern above. D, Single confocal planes with orthogonal views of MBP inside CD681 lysosomes (14 DPI). E, Full-length optic nerve section (7 DPI) immunolabeled for CD68 (top) and MBP (bottom) shows the MBP-negative area to be restricted to the region of the proximal nerve that contains the highest density of CD681 lysosomes. F, Regenerating axons (GAP431, white) are frequently associated with the area of dense phagocytic myeloid cells (CD681, red) and attenuated myelin debris (MBP1, green). G, Images of optic nerves from CR31/1 (top) and CR3/ (bottom) mice 5 DPI, immunolabeled for MBP. H, Quantitative analysis indicates that CR3/ mice have more MBP remaining near the injury site compared with littermate CR31/1 controls. I, Images of MBP- immunolabeled optic nerves from CR31/1 (top) and CR3/ (bottom) mice 5 DPI with zymosan 1 cAMP. J, Quantitative analysis demonstrates that more MBP remains in CR3/ mice com- pared with CR31/1 controls after zymosan 1 cAMP treatment. K, MBP-immunolabeled optic nerves from TPEN-treated mice 14 DPI also show increased myelin in CR3/ (bottom) vs CR31/1 (top) nerves. Scale bars: A, C, E–G, I, K, 200 mm; B, 10 mm; D, 2 mm. Graphs represent 25th to 75th percentile (box), median (line), range (whiskers), and individual data points (circles). *p . 0.05, **p , 0.01, t test.

Article Snippet: Antibodies used in immunohistochemistry experiments Antibody Target (specificity) Host species Manufacturer Dilution C1q Complement C1q (C1q) Rabbit Abcam #ab182451 1:250 C3 Complement C3 (C3) Goat MP/Cappel #55730 1:1000 C3d Complement C3d (C3d opsonin) Rabbit Dako #A0063 1:1000 CC1 CC1, APC (mature oligodendrocytes) Mouse Abcam #ab16794 1:100 CD11b Complement receptor 3, CR3, Mac1, integrin aMb 2 (monocytes, microglia, PMNs) Rat Bio-Rad #MCA711G 1:1000 CD45 PTPRC (microglia, monocytes, PMNs, NKs, lymphocytes) Rat Novus Biologicals #NB100-77417 1:500 CD68 Lysosomes (phagocytes) Rat Bio-Rad #MCA1957 1:200 CD206 Mannose receptor (monocytes, microglia) Goat R&D Systems #RB01 1:200 GAP43 Growth-associated protein 43 (regenerating axons) Sheep Custom Variable GFAP Glial fibrillary acidic protein (astrocytes) Rabbit Dako #Z0334 1:5000 GFAP Glial fibrillary acidic protein (astrocytes) Mouse Sigma #G3893 1:2500 Iba1 Ionized calcium binding adaptor molecule 1 (microglia, monocytes) Rabbit Wako #01919741 1:500 MBP Myelin basic protein (myelin) Rabbit Abcam #ab40390 1:500 Olig2 Oligodendrocyte transcription factor 2 (oligodendrocytes) Goat R&D Systems #AF2418 1:500 P2Y12 Purinergic receptor P2Y (microglia) Rabbit AnaSpec #AS-55043A 1:500 TUJ1 b -III tubulin (RGCs) Rabbit Abcam #ab18207 1:500 mean RGC count for each retina was transformed to RGC survival density (RGCs/mm2, divided by 0.235 mm2 image area), then expressed as %WT control.

Techniques: Immunolabeling

Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

Article Title: Interleukin-18 expression after focal ischemia of the rat brain: association with the late-stage inflammatory response.

doi: 10.1097/00004647-200201000-00008

Figure Lengend Snippet: FIG. 6. Confocal analysis of double-labeling immunofluores- cence for interleukin-18 (green in a) and the phagocyte marker ED1 (red in b) in ischemic lesions 3 days after cortical photo- thrombosis. (c) Sites of colocalization of both antigens appear yellow. Bars = µm.

Article Snippet: Immunofluorescent double-labeling and confocal microscopy Free-floating sections were pretreated as described previously and incubated for 16 hours at 4°C with goat polyclonal antibody against rat interleukin-18 (1:500, R&D Systems) combined with mouse monoclonal antibody ED1 against phagocytic macrophages (1:2000, Serotec, Oxford, UK).

Techniques: Labeling, Marker

Journal: Scientific Reports

Article Title: Space-like 56 Fe irradiation manifests mild, early sex-specific behavioral and neuropathological changes in wildtype and Alzheimer’s-like transgenic mice

doi: 10.1038/s41598-019-48615-1

Figure Lengend Snippet: 56 Fe irradiation reduced CD68-associated gliosis in female, but not male, APP/PS1dE9 Tg mice. ( a–d) Representative pictures of immunochemical staining for TSPO ( a ), CD68 ( b ), Iba-1 ( c ) and GFAP ( d ), respectively, on frozen brain sections of nonirradiated female Tg mice. ( e–i) %ROI quantification of immunoreactivites of TSPO in frontal cortex ( e ) and hippocampus ( f ), as well as %ROI of hippocampal CD68 ( g ), hippocampal Iba-1 ( h ), and hippocampal GFAP ( i ). CD68-positive microglia/macrophage labeling was higher in nonirradiated female Tg mice than in male Tg mice. Irradiation with 10 and 50 cGy 56 Fe reduced CD68 immuoreactivity in female, but not male, Tg mice. n = 6–9 mice/group. Mean ± SEM; # p < 0.1; *p < 0.05; ***p < 0.001.

Article Snippet: Gliosis was assessed with the following antibodies: anti-ionized calcium-binding adapter molecule 1 (Iba-1) rabbit polyclonal antibody (pAb) (a marker for all microglia/microphage, 1:500, Wako), anti-CD68 rat monoclonal antibody (mAb) (a phagocytic microglial/macrophage marker, 1:200, BD Biosciences), anti-TSPO rabbit mAb (a mitochondrial marker upregulated in neuroinflammation primarily in mouse microglia, 1:1000, Abcam), and anti- glial fibrillary acidic protein (GFAP) mouse mAb (a marker for intermediate filament proteins expressed largely in astrocytes, 1:1000; Sigma-Aldrich).

Techniques: Irradiation, Staining, Labeling

Journal: Frontiers in Aging Neuroscience

Article Title: Indomethacin Increases Neurogenesis across Age Groups and Improves Delayed Probe Trial Difference Scores in Middle-Aged Rats

doi: 10.3389/fnagi.2017.00280

Figure Lengend Snippet: Microglial activation increased with age. (A) A representative transmitted light photomicrograph of the dentate gyrus showing Iba1 + microglia (in brown). The arrows indicate Iba1 + cells that are shown in the insets under 40x. (B) A confocal image of the dentate gyrus taken under a 20x objective (with 1.5x digital zoom) showing Iba1 + cells (in blue), the marker of activation CD11b (in red), the phagocyte marker CD68 (in green) and the nuclear stain DAPI (in gray). The total number of resting Iba1 + microglia, activated Iba1 + /CD11b + microglia and phagocytic Iba1 + /CD11b + /CD68 + microglia were calculated. The arrow shows each marker independently in a phagocytic microglia. The scale bar = 50 μm. (C) shows the total number of resting (white bars), activated (gray bars), and phagocytic (black bars) microglia in vehicle-, rosiglitazone, and indomethacin-treated young, middle-aged, and aged rats. Generally, microglia numbers were higher in middle-aged and aged vs. young rats (both p < 0.01) and more resting ( p < 0.001 vs. activated and phagocytic) followed by phagocytic ( p < 0.05 vs. activated) microglia were detected. Resting microglia numbers were higher in young vs. aged rats ( p < 0.001), activated microglia numbers were higher in aged vs. young rats ( p < 0.001) and phagocytic microglia numbers higher in aged ( p < 0.001 vs. middle-aged and young rats) followed by middle-aged ( p < 0.05 vs. young rats) rats. In young and middle-aged rats, resting microglia outnumbered activated and phagocytic microglia (all p < 0.001). In aged rats, resting microglia outnumbered activated and phagocytic microglia (both p < 0.001) and phagocytic microglia outnumbered activated microglia ( p < 0.001).

Article Snippet: Sections were blocked for 20 min at RT and then incubated overnight in primary antibodies raised against (1) the immature neuronal protein doublecortin (goat anti-DCX, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and the mature neuronal protein neuronal nuclei (mouse anti-NeuN, 1:500; Chemicon, Temecula, CA), (2) the astrocyte protein glial fibrillary acidic protein (chicken anti-GFAP, 1:500; Encor Biotech, Alachua, FL) and the oligodendrocyte protein chondroitin sulfate proteoglycan (rabbit anti-NG2; Chemicon, 1:500; Temecula, CA) or (3) the microglial protein Iba1 (rabbit anti-Iba1, 1:1,000; Wako, Osaka, Japan), the activation marker CD11b (mouse anti-CD11b, 1:500; Millipore, Billerica, MA) and the phagocytic marker CD68 (goat anti-CD68, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C and then incubated in maximally cross-adsorbed fluorophore-conjugated secondary antibodies (1:500; Jackson ImmunoResearch, West Grove, PA) for 4 h at RT.

Techniques: Activation Assay, Marker, Staining