Table 1.
Summary table of optimized immunofluorescence protocols for proteins of interest.
| Antigen | Protocol Type | Retrieval | Block | Primary | Secondary |
|---|---|---|---|---|---|
|
SOX2
(anti-Sex-determining box region Y-Box 2) |
Standard | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution or 2 N HCl, 30 min at 37 °C 0.1% trypsin in PBS for 20 min at 37 °C |
5% NGS in diluent for 30 min at 37 °C | 1:50 in diluent overnight at 4 °C (Cell Signaling, 2748 S, RRID AB_823640) |
1:200 in 1XPBS for 1 hour at RT (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID AB_2338006) |
|
MSI-1
(anti-Musasahi-1) |
Standard | 2 N HCl, 30 min at 37 °C 0.1% trypsin in PBS for 20 min at 37 °C |
5% NGS in diluent for 30 min at 37 °C | 1:100 in diluent overnight at 4 °C (Millipore, Cat, MABE268, RRID AB_2576205) |
1:100 in sterile 1XPBS for 1 hour at RT (Goat anti-Mouse AlexaFluor 488, Life Technologies, A11001, RRID AB_2534069) |
|
HuCD
(anti-human neuronal protein HuC/HuD) |
Standard | 2 N HCl, 30 min at 37 °C 0.1% trypsin in PBS for 20 min at 37 °C |
5% NGS in diluent for 30 min at 37 °C | 1:10 in diluent overnight at 4 °C (Molecular Probes, Cat#16A11 RRID AB_221448) |
1:500 in sterile 1XPBS for 1 hour at RT (Goat anti-Mouse AlexaFluor 488, Life Technologies, A11001, RRID AB_2534069) |
|
GFAP
(anti-Glial Fibrillary Acidic Protein) |
Standard | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution 0.1% trypsin in PBS for 20 min at 37 °C |
5% NGS in diluent for 30 min at 37 °C | 1:400 in diluent overnight at 4 °C (Dako, Z0334, RRID AB_10013382) | 1:1000 in 1XPBS for 1 hour at RT (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID AB_2338006) |
|
Vimentin
(anti-Vimentin) |
Standard | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution 2) 0.1% trypsin in PBS for 20 min at 37 °C |
5% NGS in diluent for 30 min at 37 °C | 1:50 in diluent overnight at 4 °C (DSHB, Cat# H5, RRID AB_528506) |
1:200 in sterile 1XPBS for 1 hour at RT (Goat anti-Mouse AlexaFluor 488, Life Technologies, A11001, RRID AB_2534069) |
|
pHH3
(anti-phosphorylated Histone H3 (serine 10)) |
Abbreviated | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution | 5% NGS in 1XPBS for 1 hour at RT | 1:100 in1XPBS overnight at 4 °C (Cell Signaling, 3377 S, RRID AB_1549592) |
1:250 in 1XPBS for 1 hour at RT (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID AB_2338006) |
|
pHH3
(anti-phosphorylated Histone H3 (serine 10)) |
Immunohistochemistry | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution | 3% NGS in 1XPBS for 1 hour at RT | 1:500 in1XPBS overnight at 4 °C (Cell Signaling, 3377 S, RRID AB_1549592) |
1: 500 in 1XPBS for 1 hour at RT (Biotinylated goat anti-Rabbit, Jackson ImmunoResearch 111-066-003, RRID AB_2337966) |
|
PCNA
(anti-Proliferating Cell Nuclear Antigen) |
Abbreviated | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution | 5% NGS in 1XPBS for 1 hour at RT | 1:100 in1XPBS overnight at 4 °C (Santa Cruz BioTechnology, Inc., sc-7907) |
1:200 in 1XPBS for 1 hour at RT (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID AB_2338006) |
|
Tomato
Lectin |
Standard | 1) Citrate buffer for 12 min at 95 °C, 20 min at RT in solution 2) 0.1% trypsin in PBS for 20 min at 37 °C |
5% NGS in diluent for 30 min at 37 °C | 1:50 in diluent overnight at 4 °C (Sigma-Aldrich, Cat# L0651) |
1:500 in sterile 1XPBS for 1 hour at RT (streptavidin, AlexaFluor 488 conjugate, Life Technologies, S32354, RRID AB_2315383) |
|
VEGF
(anti-Vascular Endothelial Growth Factor A) |
Abbreviated | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution | 5% NGS in 1XPBS for 1 hour at RT | 1:50 in1XPBS overnight at 4 °C (Santa Cruz BioTechnology, Inc., sc-152, RRID AB_2212984) |
1:200 in 1XPBS for 1 hour at RT (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID AB_2338006) |
| VEGFR1 (anti-VEGF Receptor 1/Flt-1) |
Abbreviated | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution | 5% NGS in 1XPBS for 1 hour at RT | 1:100 in1XPBS overnight at 4 °C (Santa Cruz BioTechnology, Inc., sc-316, RRID AB_2107031) |
1:200 in 1XPBS for 1 hour at RT (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID AB_2338006) |
|
VEGFR2
(anti-VEGF Receptor 2/Flk-1) |
Abbreviated | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution | 5% NGS in 1XPBS for 1 hour at RT | 1:50 in1XPBS overnight at 4 °C (Santa Cruz BioTechnology, Inc., sc-6251, RRID AB_628431) |
1:200 in sterile 1XPBS for 1 hour at RT (Goat anti-Mouse AlexaFluor 488, Life Technologies, A11001, RRID AB_2534069) |
|
FGF2
(anti-basic Fibroblast Growth Factor) |
Abbreviated | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution | 5% NGS in 1XPBS for 1 hour at RT | 1:100 in1XPBS overnight at 4 °C (Santa Cruz BioTechnology, Inc., sc-79, RRID AB_631497) |
1:200 in 1XPBS for 1 hour at RT (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID AB_2338006) |
|
BrdU
(anti-5-Bromo-2′-deoxyuridine) |
Standard | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution 2) 0.1% trypsin in PBS for 20 min at 37 °C |
5% NGS in diluent for 30 min at 37 °C | 1:100 in diluent overnight at 4 °C (DSHB, G3G4, RRID AB_2618097) |
1:200 in sterile 1XPBS for 1 hour at RT (Goat anti-Mouse AlexaFluor 488, Life Technologies, A11001, RRID AB_2534069) |
|
NeuN
(anti-Neuronal Nuclei/FOX3) |
Standard | Citrate buffer for 12 min at 95 °C, 20 min at RT in solution 2) 0.1% trypsin in PBS for 20 min at 37 °C |
5% NGS in 1XPBS for 1 hour at RT | 1:125 in PBST overnight at 4 °C (Abcam, ab104225, RRID_AB10711153) |
1:1000 in 1XPBS for 1 hour at RT (Cy3-conjugated Goat anti-Rabbit IgG, 111-165-144, RRID AB_2338006) |
DSHB, Developmental Studies Hybridoma Bank, University of Iowa; min, minutes; NGS, normal goat serum; PBS, phosphate buffered saline; RT, room temperature.