Table 5.
Screening of candidate markers for human brains cells in tissue array A
| Fixation time
|
||||||
|---|---|---|---|---|---|---|
| Antigen (clone) | HIER | Dilution | 24 hr | 4 months | 10 years | Observed specificity |
| β-tubulin III | TEG | 1:500 | 4 | 3 | 0-1 | Neuropil and neuronal bodies |
| CD11b/C3bi-R | Cryo | 1:25 | 0 | 0 | 0 | No staining detected in paraffin or cryostat sections |
| CD14 | TEG | 1:25 | 3 | 0 | 0 | Perivascular macrophages |
| CD34 | TEG | 1:10 | 4 | 3 | 0 | Endothelium and white blood cells |
| CD39 | Cryo | 1:100 | 3 | 0 | 0 | Endothelium, astroglia, and macrophages |
| CD45/ LCA | TEG | 1:100 | 4 | 2-3 | 0 | Microglia, macrophages, and lymphocytes |
| CD68 (KP1) | C/Borg | 1:500 | 4 | 3 | 2 | Microglia and macrophages |
| CD68 (PGM1) | C | 1:400 | 4 | 3 | 0-1 | Lymphocytes, astrocytes, and microglia |
| CD169 | Cryo | 1:100 | 3 | 0 | 0 | Endothelial and perivascular macrophages in frozen tissue |
| CNPase | TEG/Borg | 1:300 | 4 | 3-4 | 0 | Myelinated fibers and round cell bodies |
| CNPase (RIP) | TEG/Borg | 1:100 | 4 | 4 | 0 | Myelinated fibers |
| GFAP | Non/TEG | 1:2000 | 4 | 3-4 | 3 | Astroglia in WM and neocortex |
| HLA-DR | TEG/TRS | 1:15 | 3-4 | 2 | 0 | Microglia/macrophages and lymphocytes in adult tissue |
| Ki67-antigen | TEG | 1:50 | 4 | 4 | 2-3 | Single or double profiles in perivascular space and SVZ |
| MAP-2 | TEG | 1:500 | 3-4 | 2-3 | 0 | Neurons and proximal part of apical dendrites |
| MBP | TEG/Borg | 1:300 | 4 | 3-4 | 3 | Myelinated fibers in adult. |
| Nestin | TEG/TRS | 1:100 | 4 | 3 | 2 | Endothelial cells/vessel wall |
| NeuN | TEG/TRS | 1:400 | 3-4 | 0 | 0 | Neuronal cell bodies |
| Neurofilament (2F11) | TEG | 1:200 | 4 | 4 | 4 | Neuronal cell processes |
| Neurofilament (DA2/FNP7/RmdO20.11) | TEG | 1:500 | 3-4 | 3 | 0-1 | Neuronal soma, apical dendrites and axons |
| NG2 | Cryo | 1:300 | 0 | 0 | 0 | No staining in neither paraffin nor cryostat sections |
| Nkx-2.2 | TEG/Borg | 1:50 | 3 | 0 | 0 | No reaction in adult tissue. Small cells in WM in newborn tissue |
| NSE | TEG | 1:1000 | 3-4 | 3-4 | 2 | Neuropil |
| O4 sulfatide | TEG/TRS | 1:80 | 1-2 | 1 | 0 | Myelinated fibers |
| PDGFα-R | TEG | 1:300 | 4-3 | 0 | 0 | No reaction in adult tissue. Small cells in WM in newborn tissue |
| p25α-antigen | TEG | 1:1000 | 4 | 4 | 0 | Neuropil and round cell bodies connected to myelin fibers |
| S100β protein | Non/TEG | 1:500 | 4 | 4 | 3 | Astroglia in WM and neocortex |
| TOAD-64 | TEG/C | 1:1000 | 1 | 0 | 0 | Neuropil |
| Vimentin | TEG | 1:200 | 4 | 2 | 1-3 | Astroglia and endothelial cells/vessel wall |
Each antibody was applied in a range of dilutions using different epitope retrieval methods in sections from tissue array A (Table 1). Staining results of repeated experiments were scored, and on this basis, the optimal staining result for each antibody was identified. For all antibodies, HIER was superior to proteolytic pretreatment, but the buffer of choice varied between antibodies, as did dilutions of antibodies. HIER, heat-induced epitope retrieval; Borg, Borg Decloak epitope retrieval buffer; C, citrate-buffer, pH 6.0; Cryo, staining in cryostat sections with no epitope retrieval; Non, no epitope retrieval; TEG, Tris-EGTA buffer, pH 9.0; TRS, target retrieval solution; SVZ, subventricular zone; WM, white matter.