Antibodies used / Provider / Use / Fixation (Volume/Medium) / Fixation time/temperature / Washes (Number/Volume/Medium) / Permeabilization (Volume, medium) / Permeabilization time/temperature / Washes (Number/Volume/Medium) / Staining (Volume/Medium) / Staining time/temperature / Ab. Amount used / sample / Staining sequence, if any / Post stain wash (Number/Volume/Medium)
Anti-human STAT1 (N-term.), PE, Mouse IgG1, k / BD / Detecting ab. / 1 ml 1.5% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / None (concomitant stains) / 1x / 2 ml / WB
Isotype control, PE, Mouse IgG1, k / BD / Isotype cont. / 1 ml 1.5% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / None (concomitant stains) / 1x / 2 ml / WB
Anti-human STAT1 pY701, AF647, Mouse IgG2a / BD / Detecting ab. / 1 ml 1.5% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / None (concomitant stains) / 1x / 2 ml / WB
Isotype control, AF647, Mouse IgG2a, k / BD / Isotype cont. / 1 ml 1.5% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 0.3 ul / 1x10E+6 cells / None (concomitant stains) / 1x / 2 ml / WB
Abbreviations:
PFA; Paraformaldehyde
RT; Room temperature
WB; Standard bovine serum albumine-supplemented flow cytometric wash buffer
STAT2Antibodies used / Provider / Use / Fixation (Volume/
medium) / Fixation time/temp
erature / Washes (Number/Volu
me/Medium) / Permeabilization
(Volume/medium) / Permeabilization time/temperature / Washes (Number/Volu
me/Medium) / Staining (Volume/Medi
um) / Staining time/temper
ature / Ab. Amount used / sample / Staining sequence, if any / Post/between stain washes (Number/Volu
me/Medium)
Anti-human STAT2 unconjugated, Rabbit IgG / Cell Signaling / Primary ab. / 1 ml 4.0% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 2 ul / 1x10E+6 cells / 1st stainig cycle / 1x / 2 ml / WB
Anti-rabbit IgG, APC, Goat IgG / R&D / Secondary ab. / 1 ml 4.0% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / +4C / 10 ul / 1x10E+6 cells / 2nd staining cycle / 1x / 2 ml / WB
Anti-human STAT2 pY689 FITC, Rabbit IgG / R&D / Detecting ab. / 1 ml 4.0% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / 3d staining cycle / 1x / 2 ml / WB
Isotype control, FITC, Rabbit IgG / R&D / Isotype cont. / 1 ml 4.0% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / 3d staining cycle / 1x / 2 ml / WB
Abbreviations:
PFA; Paraformaldehyde
RT; Room temperature
WB; Standard bovine serum albumine-supplemented flow cytometric wash buffer
STAT3Antibodies used / Provider / Use / Fixation (Volume/
medium) / Fixation time/temp
erature / Washes (Number/Volu
me/Medium) / Permeabilization
(Volume/medium) / Permeabilization time/temperature / Washes (Number/Volu
me/Medium) / Staining (Volume/Medi
um) / Staining time/temper
ature / Ab. Amount used / sample / Staining sequence, if any / Post stain wash (Number/Volu
me/Medium)
Anti-human STAT3, PE, Mouse IgG1,l / BD / Detecting ab. / 1 ml 1.5% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / None (concomitant stains) / 1x / 2 ml / WB
Isotype control, PE, Mouse IgG1, k / BD / Isotype cont. / 1 ml 1.5% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / None (concomitant stains) / 1x / 2 ml / WB
Anti-human STAT3 pY705, AF647, Mouse IgG 2b,k / BD / Detecting ab. / 1 ml 1.5% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / None (concomitant stains) / 1x / 2 ml / WB
Isotype control, AF647, Mouse IgG2a, k / BD / Isotype cont. / 1 ml 1.5% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 0.3 ul / 1x10E+6 cells / None (concomitant stains) / 1x / 2 ml / WB
Abbreviations:
PFA; Paraformaldehyde
RT; Room temperature
WB; Standard bovine serum albumine-supplemented flow cytometric wash buffer
STAT4Antibodies used / Provider / Use / Fixation (Volume/
medium) / Fixation time/temp
erature / Washes (Number/Volu
me/Medium) / Permeabilization
(Volume/medium) / Permeabilization time/temperature / Washes (Number/Volu
me/Medium) / Staining (Volume/Medi
um) / Staining time/temper
ature / Ab. Amount used / sample / Staining sequence, if any / Post/between stain washes (Number/Volu
me/Medium)
Anti-human STAT4, unconjugated, Rabbit IgG / Invitrogen / Primary ab. / 1 ml 10% Reagent A / 15 min / RT / - / 3 ml 100% Methanol, prechilled to -20C / 10 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / Reagent B / 30 min / RT / 4 ul / 1x10E+6 cells / 1st stainig cycle / 1x / 2 ml / WB
Goat anti-rabbit IgG-FITC / Invitrogen / Secondary ab. / 1 ml 10% Reagent A / 15 min / RT / - / 3 ml 100% Methanol, prechilled to -20C / 10 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / Reagent B / 30 min / RT / 10 ul / 1x10E+6 cells / 2nd stainig cycle / 1x / 2 ml / WB
Anti-human STAT4 pY693, AF647, Mouse IgG 2b,k / BD / Detecting ab. / 1 ml 10% Reagent A / 15 min / RT / - / 3 ml 100% Methanol, prechilled to -20C / 10 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / Reagent B / 30 min / RT / 20 ul / 1x10E+6 cells / 1st stainig cycle / 1x / 2 ml / WB
Isotype control, AF647, Mouse IgG2a, k / BD / Isotype cont. / 1 ml 10% Reagent A / 15 min / RT / - / 3 ml 100% Methanol, prechilled to -20C / 10 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / Reagent B / 30 min / RT / 20 ul / 1x10E+6 cells / 1st stainig cycle / 1x / 2 ml / WB
Abbreviations and uncommon reagents:
PFA; Paraformaldehyde
RT; Room temperature
WB; Standard bovine serum albumine-supplemented flow cytometric wash buffer
Reagent A - Permeabilization Medium (Invitrogen)
Reagent B -Fixation Medium (Invitrogen)
STAT5Antibodies used / Provider / Use / Fixation (Volume/
medium) / Fixation time/temp
erature / Washes (Number/Volu
me/Medium) / Permeabilization
(Volume/medium) / Permeabilization time/temperature / Washes (Number/Volu
me/Medium) / Staining (Volume/Medi
um) / Staining time/temper
ature / Ab. Amount used / sample / Staining sequence, if any / Post/between stain washes (Number/Volu
me/Medium)
Anti-human STAT5 unconjugated, Rabbit IgG / Santa Cruz / 1st ab / 1 ml 4.0% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 2 ul / 1x10E+6 cells / 1st stainig cycle / 1x / 2 ml / WB
Anti-rabbit IgG, APC, Goat IgG / R&D / 2nd ab / 1 ml 4.0% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / +4C / 10 ul / 1x10E+6 cells / 2nd staining cycle / 1x / 2 ml / WB
Anti-human STAT5 pY694 AF488, Mouse IgG1 / BD / Detecting ab. / 1 ml 4.0% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / 3d staining cycle / 1x / 2 ml / WB
Isotype control AF488, Mouse IgG 1k / BD / Isotype cont. / 1 ml 4.0% PFA / 10 min / RT / 1x / 2 ml / WB / 1 ml 100% Methanol, prechilled to -20C / 20 min - overnight / -20C / 2x / 2 ml / WB / 100 ul / WB / 30 min / RT / 20 ul / 1x10E+6 cells / 3d staining cycle / 1x / 2 ml / WB
Abbreviations:
PFA; Paraformaldehyde
RT; Room temperature
WB; Standard bovine serum albumine-supplemented flow cytometric wash buffer