Incubation:Article Title: Complex I inhibition combined with TLR activation in the breast tumor microenvironment educates cytotoxic neutrophils
Article Snippet: ROIs were set on mammary glands and the maximal peak of the luminescent signals over the five readings were used for analysis. pNFB Flow Cytometry Purified Ly6G+ cells were immediately fixed with as per manufacturers protocol with FoxP3 fixation/permeabilization kit (00-5523-00, Thermofisher). .. 1 x 105 cells were washed with diluted permeabilization buffer and incubated with 1:100 of pNF-B antibody (93H1, CST) for 1 hour at RT. .. Cells were washed and incubated with 1:500 goat anti-rabbit Alexa Fluor 488 (A-11008, Invitrogen) for 30 minutes at RT.
Article Title: HBV capsid assembly modulators differentially modulate the assembly of wild-type and drug-resistant core protein chimeric nucleocapsids and empty capsids
Article Snippet: .. At 6 h post transfection, the cells were mock-treated with control solvent (DMSO) or GLS4 (10 μM) for 48 h. The cells were fixed with 95% methanol and 5% glacial acetic acid and then washed 3 times with 1 × PBS followed by 1 h of incubation with the blocking and permeabilization buffer (5% BSA, 10% FBS, 0.3% Triton-X-100 in 1 × PBS, all (vol/vol)) at room temperature for 1 h. Thereafter, cells were incubated with an anti-HA antibody (1:100 dilution, Cat. NO. 3724S, Cell Signaling Technology, MA) in the dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at 4 °C overnight, washed for 3 times and incubated with an Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (1:1000 dilution, Cat. NO. A-11034, Invitrogen, MA). .. The nuclei were visualized by DAPI (1 μg/mL) in dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at room temperature for 1 h. After 3 times of washing, the cover slides with cells were then transferred onto a glass slide with 2 μl ProLong Gold antifade solution (Cat. NO. P36930 , Thermo Fisher Scientific, Waltham, MA) and sealed with CoverGrip Coverslip Sealant (Cat. NO. 23005, Biotium Inc., Fremont, CA).
Article Title: CRAMP1-dependent histone H1 biogenesis is essential for topoisomerase II inhibitor tolerance
Article Snippet: .. Briefly, cells were permeabilized with permeabilization buffer (20 mM HEPES-KOH, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 0.1% Triton X-100) containing protease inhibitors (Sigma) for 15 min at RT and incubated with indicated antibodies (diluted to 0.75 μg/μl in permeabilization buffer) for 1 h. Antibody solution was removed and pAG-MNase (Cell Signaling; diluted 1:33 in permeabilization buffer) was added for 1 h at RT. ..
Article Title: Single-cell quantification of senescence burden reveals cell type-specific ageing dynamics across organs
Article Snippet: .. The samples were incubated with Donkey Serum (Jackson ImmunoResearch Laboratories) in permeabilization buffer for 5 minutes, and stained with the 3SMs, anti-p16 AF647 (1:1000, Abcam, ab192054), anti-γH2.ax PE (1:1000, BD Biosciences, 562377) and anti-Bcl-xl PE-Cy7 (1:1000, Cell Signaling Technology, 81965) (30 minutes at 4°C). ..
Article Title: EZH2-TTP-mTORC1 Axis Drives Phenotypic Plasticity and Therapeutic Vulnerability in Lethal Prostate Cancer
Article Snippet: .. After two washes with permeabilization buffer, cells were resuspended in FACS buffer containing antibody against Zfp36 (LS-B5606, LSBio), TSC2 (D93F12, Cell Signaling), p-S6K (9205, Cell Signaling), and p4E-BP1 (2855, Cell Signaling) and incubated 30 minutes at room temperature. .. After two washes cells were resuspended in FACS buffer containing the secondary antibody (Biolegend 406410).
Article Title: Downregulation of AdipoR1 in the hippocampus impairs synaptic function and structure and causes depression-like behavior
Article Snippet: .. One randomly selected set of brain sections was washed128 with 0.01 M PBS and permeabilization buffer (0.3% Tween-20 in PBS) for 30129 min, immersed in blocking buffer containing 5% goat serum, 1% bovine serum130 albumin and 0.3% Tween-20 in PBS at 37°C for 2 h and then incubated with131 primary antibodies (rabbit anti-spinophilin, CST, Cat #14136) at 4°C for 72 h.132 The sections were then incubated with secondary antibodies (Electron133 Microscopy Sciences, ImmunoGold Reagents, goat anti-rabbit IgG) at 37°C for134 3 h. Next, the sections were postfixed in the dark with 2% glutaraldehyde for135 10 min. .. Subsequently, the sections were subjected to silver enhancement136 (Aurion R-Gent Silver Enhancement Kit, Electron Microscopy Sciences) at137 25°C for 25 min and washed with deionized water followed by 0.01 M PBS.138 After being stained with hematoxylin for 1 min, the sections were dehydrated in139 gradient alcohol, cleared with xylene and sealed with neutral gum.140 For immunofluorescence, the brain sections were equilibrated in a series141 of sucrose gradients for dehydration at 4°C (10%, 24 h; 20%, 24 h; and 30%,142 48 h).
Transfection:Article Title: HBV capsid assembly modulators differentially modulate the assembly of wild-type and drug-resistant core protein chimeric nucleocapsids and empty capsids
Article Snippet: .. At 6 h post transfection, the cells were mock-treated with control solvent (DMSO) or GLS4 (10 μM) for 48 h. The cells were fixed with 95% methanol and 5% glacial acetic acid and then washed 3 times with 1 × PBS followed by 1 h of incubation with the blocking and permeabilization buffer (5% BSA, 10% FBS, 0.3% Triton-X-100 in 1 × PBS, all (vol/vol)) at room temperature for 1 h. Thereafter, cells were incubated with an anti-HA antibody (1:100 dilution, Cat. NO. 3724S, Cell Signaling Technology, MA) in the dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at 4 °C overnight, washed for 3 times and incubated with an Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (1:1000 dilution, Cat. NO. A-11034, Invitrogen, MA). .. The nuclei were visualized by DAPI (1 μg/mL) in dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at room temperature for 1 h. After 3 times of washing, the cover slides with cells were then transferred onto a glass slide with 2 μl ProLong Gold antifade solution (Cat. NO. P36930 , Thermo Fisher Scientific, Waltham, MA) and sealed with CoverGrip Coverslip Sealant (Cat. NO. 23005, Biotium Inc., Fremont, CA).
Control:Article Title: HBV capsid assembly modulators differentially modulate the assembly of wild-type and drug-resistant core protein chimeric nucleocapsids and empty capsids
Article Snippet: .. At 6 h post transfection, the cells were mock-treated with control solvent (DMSO) or GLS4 (10 μM) for 48 h. The cells were fixed with 95% methanol and 5% glacial acetic acid and then washed 3 times with 1 × PBS followed by 1 h of incubation with the blocking and permeabilization buffer (5% BSA, 10% FBS, 0.3% Triton-X-100 in 1 × PBS, all (vol/vol)) at room temperature for 1 h. Thereafter, cells were incubated with an anti-HA antibody (1:100 dilution, Cat. NO. 3724S, Cell Signaling Technology, MA) in the dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at 4 °C overnight, washed for 3 times and incubated with an Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (1:1000 dilution, Cat. NO. A-11034, Invitrogen, MA). .. The nuclei were visualized by DAPI (1 μg/mL) in dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at room temperature for 1 h. After 3 times of washing, the cover slides with cells were then transferred onto a glass slide with 2 μl ProLong Gold antifade solution (Cat. NO. P36930 , Thermo Fisher Scientific, Waltham, MA) and sealed with CoverGrip Coverslip Sealant (Cat. NO. 23005, Biotium Inc., Fremont, CA).
Solvent:Article Title: HBV capsid assembly modulators differentially modulate the assembly of wild-type and drug-resistant core protein chimeric nucleocapsids and empty capsids
Article Snippet: .. At 6 h post transfection, the cells were mock-treated with control solvent (DMSO) or GLS4 (10 μM) for 48 h. The cells were fixed with 95% methanol and 5% glacial acetic acid and then washed 3 times with 1 × PBS followed by 1 h of incubation with the blocking and permeabilization buffer (5% BSA, 10% FBS, 0.3% Triton-X-100 in 1 × PBS, all (vol/vol)) at room temperature for 1 h. Thereafter, cells were incubated with an anti-HA antibody (1:100 dilution, Cat. NO. 3724S, Cell Signaling Technology, MA) in the dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at 4 °C overnight, washed for 3 times and incubated with an Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (1:1000 dilution, Cat. NO. A-11034, Invitrogen, MA). .. The nuclei were visualized by DAPI (1 μg/mL) in dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at room temperature for 1 h. After 3 times of washing, the cover slides with cells were then transferred onto a glass slide with 2 μl ProLong Gold antifade solution (Cat. NO. P36930 , Thermo Fisher Scientific, Waltham, MA) and sealed with CoverGrip Coverslip Sealant (Cat. NO. 23005, Biotium Inc., Fremont, CA).
Blocking Assay:Article Title: HBV capsid assembly modulators differentially modulate the assembly of wild-type and drug-resistant core protein chimeric nucleocapsids and empty capsids
Article Snippet: .. At 6 h post transfection, the cells were mock-treated with control solvent (DMSO) or GLS4 (10 μM) for 48 h. The cells were fixed with 95% methanol and 5% glacial acetic acid and then washed 3 times with 1 × PBS followed by 1 h of incubation with the blocking and permeabilization buffer (5% BSA, 10% FBS, 0.3% Triton-X-100 in 1 × PBS, all (vol/vol)) at room temperature for 1 h. Thereafter, cells were incubated with an anti-HA antibody (1:100 dilution, Cat. NO. 3724S, Cell Signaling Technology, MA) in the dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at 4 °C overnight, washed for 3 times and incubated with an Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (1:1000 dilution, Cat. NO. A-11034, Invitrogen, MA). .. The nuclei were visualized by DAPI (1 μg/mL) in dilution buffer (1% BSA, 1% FBS, 0.3% Triton-X-100 in 1 × PBS) at room temperature for 1 h. After 3 times of washing, the cover slides with cells were then transferred onto a glass slide with 2 μl ProLong Gold antifade solution (Cat. NO. P36930 , Thermo Fisher Scientific, Waltham, MA) and sealed with CoverGrip Coverslip Sealant (Cat. NO. 23005, Biotium Inc., Fremont, CA).
Article Title: Downregulation of AdipoR1 in the hippocampus impairs synaptic function and structure and causes depression-like behavior
Article Snippet: .. One randomly selected set of brain sections was washed128 with 0.01 M PBS and permeabilization buffer (0.3% Tween-20 in PBS) for 30129 min, immersed in blocking buffer containing 5% goat serum, 1% bovine serum130 albumin and 0.3% Tween-20 in PBS at 37°C for 2 h and then incubated with131 primary antibodies (rabbit anti-spinophilin, CST, Cat #14136) at 4°C for 72 h.132 The sections were then incubated with secondary antibodies (Electron133 Microscopy Sciences, ImmunoGold Reagents, goat anti-rabbit IgG) at 37°C for134 3 h. Next, the sections were postfixed in the dark with 2% glutaraldehyde for135 10 min. .. Subsequently, the sections were subjected to silver enhancement136 (Aurion R-Gent Silver Enhancement Kit, Electron Microscopy Sciences) at137 25°C for 25 min and washed with deionized water followed by 0.01 M PBS.138 After being stained with hematoxylin for 1 min, the sections were dehydrated in139 gradient alcohol, cleared with xylene and sealed with neutral gum.140 For immunofluorescence, the brain sections were equilibrated in a series141 of sucrose gradients for dehydration at 4°C (10%, 24 h; 20%, 24 h; and 30%,142 48 h).
Staining:Article Title: Single-cell quantification of senescence burden reveals cell type-specific ageing dynamics across organs
Article Snippet: .. The samples were incubated with Donkey Serum (Jackson ImmunoResearch Laboratories) in permeabilization buffer for 5 minutes, and stained with the 3SMs, anti-p16 AF647 (1:1000, Abcam, ab192054), anti-γH2.ax PE (1:1000, BD Biosciences, 562377) and anti-Bcl-xl PE-Cy7 (1:1000, Cell Signaling Technology, 81965) (30 minutes at 4°C). ..
Article Title: The type-I interferon priming signal balances antibacterial and antitumor trained immunity in alveolar macrophages.
Article Snippet: Tissue-resident macrophages may be trained to confer an enhanced response to heterologous restimulation and thus foster versatile trained immunity (TI) against both infections and tumors.. However, the key priming signals that contribute to such functional versatility in trained macrophages are not fully understood.. Here, we show that influenza A virus (IAV) infection in mice induces lasting transcriptional imprints of acute type-I interferon (IFN-I) signaling in lung-resident alveolar macrophages (AMs) that confer balanced antibacterial and antitumor TI responses.
Transferring:
FACS:Article Title: EZH2-TTP-mTORC1 Axis Drives Phenotypic Plasticity and Therapeutic Vulnerability in Lethal Prostate Cancer
Article Snippet: .. After two washes with permeabilization buffer, cells were resuspended in FACS buffer containing antibody against Zfp36 (LS-B5606, LSBio), TSC2 (D93F12, Cell Signaling), p-S6K (9205, Cell Signaling), and p4E-BP1 (2855, Cell Signaling) and incubated 30 minutes at room temperature. .. After two washes cells were resuspended in FACS buffer containing the secondary antibody (Biolegend 406410).
Microscopy:Article Title: Downregulation of AdipoR1 in the hippocampus impairs synaptic function and structure and causes depression-like behavior
Article Snippet: .. One randomly selected set of brain sections was washed128 with 0.01 M PBS and permeabilization buffer (0.3% Tween-20 in PBS) for 30129 min, immersed in blocking buffer containing 5% goat serum, 1% bovine serum130 albumin and 0.3% Tween-20 in PBS at 37°C for 2 h and then incubated with131 primary antibodies (rabbit anti-spinophilin, CST, Cat #14136) at 4°C for 72 h.132 The sections were then incubated with secondary antibodies (Electron133 Microscopy Sciences, ImmunoGold Reagents, goat anti-rabbit IgG) at 37°C for134 3 h. Next, the sections were postfixed in the dark with 2% glutaraldehyde for135 10 min. .. Subsequently, the sections were subjected to silver enhancement136 (Aurion R-Gent Silver Enhancement Kit, Electron Microscopy Sciences) at137 25°C for 25 min and washed with deionized water followed by 0.01 M PBS.138 After being stained with hematoxylin for 1 min, the sections were dehydrated in139 gradient alcohol, cleared with xylene and sealed with neutral gum.140 For immunofluorescence, the brain sections were equilibrated in a series141 of sucrose gradients for dehydration at 4°C (10%, 24 h; 20%, 24 h; and 30%,142 48 h).
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