Table 1.
Summary table of optimized immunofluorescent protocols for proteins of interest.
| Antigen | Retrieval | Block | Primary | Secondary |
|---|---|---|---|---|
| BrdU (Anti-5-bromo-2′-deoxyuridine) |
Citrate buffer 12 min at 95 °C, 20 min at room temperature in solution 0.1% trypsin in PBS 20 min at 37 °C |
5% NGS in diluent 30 min at 37 °C | 1:100 in diluent overnight at 4 °C (DSHB, mouse monoclonal, G3G4, RRID: AB_2618097) |
1:200 in sterile 1XPBS for 1 h at room temperature (Goat anti-Mouse AlexaFluor 488, Life Technologies, A11001, RRID: AB_2534069) |
| GFAP (Anti-glial fibrillary acidic protein) |
Citrate buffer 12 min at 95 °C, 20 min at room temperature in solution 0.1% trypsin in PBS 20 min at 37 °C |
5% NGS in diluent 30 min at 37 °C | 1:400 in diluent overnight at 4 °C (DAKO, rabbit polyclonal, Z0334, RRID: AB_10013382) | 1:1000 in 1XPBS for 1 h at room temperature (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID: AB_2338006) |
| HuCD (Anti-human neuronal protein HuC/HuD) |
Tris buffer 30 min at 95 °C, 30 min at room temperature in solution | 10% NGS in 0.3% Triton-X-100 in 1XPBS 30 min at room temperature | 1:10 in 1%BSA in 1XPBS overnight at 4 °C (Molecular Probes, mouse monoclonal, 16A11, RRID: AB_221448) |
1:500 in sterile 1XPBS for 1 h at room temperature (Goat anti-Mouse AlexaFluor 488, Life Technologies, A11001, RRID: AB_2534069) |
| NeuN (Anti-neuronal nuclei) |
Tris buffer 30 min at 95 °C, 30 min at room temperature in solution | 10% NGS in 0.3% Triton-X-100 in 1XPBS 30 min at room temperature | 1:500 in 1%BSA in 1XPBS overnight at 4 °C (Abcam, rabbit polyclonal, ab104225, RRID: AB_10711153) |
1:1000 in 1XPBS for 1 h at room temperature (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID: AB_2338006) |
| PCNA (Anti-proliferating cell nuclear antigen) |
none | 3% NGS in 1XPBS 1 h at room temperature | 1:100 in1XPBS overnight at 4 °C (Santa Cruz Biotech., rabbit polyclonal, sc-7907, RRID: AB_2160375) |
1:200 in 1XPBS for 1 h at room temperature (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID: AB_2338006) |
| pHH3 (Anti-phospho-histone H3 (Ser10)) |
Citrate buffer 12 min at 95 °C, 20 min at room temperature in solution | 3% NGS in 1XPBS 1 h at room temperature | 1:100 in1XPBS overnight at 4 °C (Cell Signaling, rabbit polyclonal, 3377S, RRID: AB_1549592) |
1:250 in 1XPBS for 1 h at room temperature (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID: AB_2338006) |
| SOX2 (Anti-SRY (sex-determining box region Y) box2) |
Citrate buffer 12 min at 95 °C, 20 min at room temperature in solution 0.1% trypsin in PBS 20 min at 37 °C |
5% NGS in diluent 30 min at 37 °C | 1:50 in diluent overnight at 4 °C (Cell Signaling, rabbit polyclonal, 2748S, RRID: AB_823640) |
1:200 in 1XPBS for 1 h at room temperature (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID: AB_2338006) |
| Vimentin (Anti-Vimentin) |
Citrate buffer 12 min at 95 °C, 20 min at room temperature in solution 0.1% trypsin in PBS 20 min at 37 °C |
5% NGS in diluent 30 min at 37 °C | 1:50 in diluent overnight at 4 °C (DSHB, mouse monoclonal, H5, RRID: AB_528506) |
1:200 in sterile 1XPBS for 1 h at room temperature (Goat anti-Mouse AlexaFluor 488, Life Technologies, A11001, RRID: AB_2534069) |
BSA, bovine serum albumin; DSHB, Developmental Studies Hybridoma Bank, University of Iowa, USA; NGS, normal goat serum; PBS, phosphate buffered saline; RRID, Research Resource Identifier.