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[IF=6.014] Lucca LE et al. TIGIT signaling restores suppressor function of Th1 Tregs. JCI Insight. 2019 Feb 7;4(3). pii: 124427.  FCM ;  Human.  

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[IF=2.81] Kinoshita et al. Associations between Forkhead Box O1 (FoxO1) Expression and Indicators of Hepatic Glucose Production in Transition Dairy Cows Supplemented with Dietary Nicotinic Acid. (2016) PLoS.On. 11:e0146670  WB ;  Bovine.  

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[IF=2.16] Li, Xinxin, et al. "Long-term thermal manipulation in the late incubation period can inhibit breast muscle development by activating endoplasmic reticulum stress in duck (Anasplatyrhynchos domestica)." Journal of thermal biology 70.Pt B (2017): 37.  WB ;  Other Species.  

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Paraformaldehyde-fixed, paraffin embedded (Rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat placenta); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse liver); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat colon); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse colon); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse testis); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat testis); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human gastric carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat lung); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-FoxO1 (Ser256)) Polyclonal Antibody, Unconjugated (bs-3142R ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Black line : Positive blank control (Hela); Negative blank control (Molt4）
Green line : Primary Antibody (Rabbit Anti-Phospho-FoxO1 (Ser256) antibody (bs-3142R) )
Orange line：Isotype Control Antibody (Rabbit IgG) .
Blue line : Secondary Antibody (Goat anti-rabbit IgG-AF488)
Hela（Positive）and Molt4（Negative control）cells (black) were fixed with 4% PFA for 10min at room temperature, permeabilized with 90% ice-cold methanol for 20 min at -20℃, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with Phospho-FoxO1 (Ser256) Antibody(bs-3142R)at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).

Blank control: HepG2.
Primary Antibody (green line): Rabbit Anti-Phospho-FoxO1 (Ser256) antibody (bs-3142R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.

Blank control(blue): Hela(fixed with 2% paraformaldehyde (10 min) and then permeabilized with ice-cold 90% methanol for 30 min on ice).
Primary Antibody: Rabbit Anti- Phospho-FoxO1 (Ser256)/FITC antibody(bs-3142R-FITC), Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA;
Isotype Control Antibody: Rabbit IgG/FITC(orange) ,used under the same conditions.

phosphopeptide non phosphopeptide