Cisplatin was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Cell counting kit-8 (CCK-8; C0037) and 4′,6-diamidino-2-phenylindole (DAPI; C1005) were purchased from the Beyotime Institute of Biotechnology (Haimen, China) and stored at −20°C. Antibodies including non-phospho (active) β-catenin (Ser33/37/Thr41) (D13A1) rabbit mAb (#8814), SAPK/JNK rabbit mAb (#9252) and CaMKII (pan) (D11A10) Rabbit mAb (#4436) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), and the β-actin mouse mAb (JT9001S) was purchased from Anbo Biotechnology Co., Ltd, (San Francisco, CA, USA). The Wnt signaling inhibitor (XAV-939) and activator (CHIR-99021) were purchased from the Selleck Chemicals (Shanghai, China), and completely dissolved in DMSO at a stock concentration (1 mM). Fluorescein isothiocyanate (FITC)-Conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (ZF-0312) and FITC-Conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (ZF-0315) were from OriGene Technologies, Inc. (Beijing, China).
4 6 diamidino 2 phenylindole dapi c1005
Manufactured by Beyotime
Sourced in China
4',6-diamidino-2-phenylindole (DAPI; C1005) is a fluorescent stain that binds strongly to DNA. It is commonly used in biological research for the detection and visualization of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Cisplatin, by Merck Group (1 mentions)
Confocal microscope, by Nikon (1 mentions)
P0099, by Beyotime (1 mentions)
Ab6326, by Abcam (1 mentions)
B5002, by Merck Group (1 mentions)
Ab150107, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Abcam (1 mentions)
Sc 28338, by Santa Cruz Biotechnology (1 mentions)
Alexafluor 647 donkey anti mouse igg, by Abcam (1 mentions)
P1585, by Merck Group (1 mentions)
Ab150073, by Abcam (1 mentions)
Anti β actin antibody sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab2732, by Abcam (1 mentions)
Ab32505, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit or mouse secondary antibodies, by Merck Group (1 mentions)
3 3 dioctadecyloxacarbocyanine perchlorate dio c1993s, by Beyotime (1 mentions)
7 protocols using 4 6 diamidino 2 phenylindole dapi c1005
1
Wnt Signaling Pathway Modulation
2
Immunofluorescence Staining of Frozen Liver Sections
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Frozen liver sections were fixed with 4% paraformaldehyde and incubated with 0.25% Triton X-100 at room temperature for 10 min. For nucleoprotein like Gli-1, mixture of glacial acetic acid and ethanol (volume/volume 1:3) was used to increase the permeability of primary antibodies into nuclear membrane. Sections were blocked with 5% BSA at room temperature for 30 min and incubated with primary antibodies in 1% BSA at 4 °C overnight. On the second day, sections were incubated with secondary antibody at 37 °C for 1 h, and nucleus was counter-stained with 4'6-diamidino-2-phenylindole (DAPI) (C1005; Beyotime, Shanghai, China). Cells in 5 randomly-selected fields at 630× under a confocal microscope (Nikon Corporation, Tokyo, Japan) were used to quantify with Image J software for each section. Commercial sources of all antibodies used in this study are listed inTable S2
3
Immunofluorescence Staining of HSCs and LX-2 Cells
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After immobilizing with 4% (V/V) paraformaldehyde (P0099; Beyotime) and a PBS wash, primary HSCs and LX-2 cells were permeabilized using 0.5% (V/V) Triton X-100 and blocked with 5% (V/V) bovine serum albumin (BSA) for 30 min. They were then treated with primary antibodies, including anti-α-SMA (ab124964), anti-Col1A1 (ab283694), anti-BECN1 (ab62557), and anti-SLC7A11 (ab307601). After this, cells were stained with secondary antibodies, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (C1005; Beyotime). The prepared samples were visualized under a microscope (Nikon, Tokyo, Japan).
4
Quantitative Analysis of Cell Proliferation
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Cells were seeded on coverslips, and incubated with 25 μM BrdU (B5002, Sigma-Aldrich, MO, USA) for 40 min, then washed with PBS and fixed in 4% paraformaldehyde (PFA) for 20 min. Subsequently, cells were treated with 1 M HCl and blocked with 10% goat serum for 1 h, and then incubated with BrdU antibody (ab6326, Abcam, MA, USA) overnight at 4 °C followed by Alexa FluorR 488 goat anti-rat IgG secondary antibody (H + L) (A-11006, Thermo Fisher, IL, USA) for 2 h at 25 °C. Nuclear was stained with 300 nM 4′, 6-diamidino-2-phenylindole (DAPI) (C1005, Beyotime, Shanghai, China) for 15 min at 25 °C. The percentage of BrdU-positive cells was calculated and 6 microscopic fields were counted (Nikon 80i, Nikon Corporation, Tokyo, Japan).
5
Immunofluorescent Labeling of Decidual Tissue and THP-1 Macrophages
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For labeling, decidual tissue sections were incubated with primary antibodies: mouse monoclonal anti-SGK1 antibodies (1:50, SC-28338, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-CD68 antibodies (1:500, 76437, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The slides of THP-1 cells before and after phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, P1585) pretreatment were incubated with anti-CD68 antibodies at 4°C overnight. Then, the sections of human decidual tissue and THP-1 macrophages were incubated with Alexa Fluor® 488 Donkey Anti-Rabbit IgG (1:400, ab150073, Abcam, Cambridge, UK) and Alexa Fluor® 647 Donkey Anti-Mouse IgG (1:400, ab150107, Abcam, UK) secondary antibodies at 37°C for 1 h in the dark. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, C1005, Beyotime Biotechnology, Shanghai, China) and washed using PBS (3 × 5 min). The slices were photographed and results were recorded using a laser confocal microscope (LSM 710, Zeiss, Wetzlar, Germany).
6
Autophagy Regulation by Rapamycin
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Rapamycin (R0935), rabbit polyclonal anti-LC3B antibody (L7543) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit or -mouse secondary antibodies were purchased from Sigma-Aldrich (St. Louis, USA). Rabbit polyclonal anti-ATG5 antibody (sc-33210) and mouse monoclonal anti-β-actin antibody (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Rabbit anti-p-AKT (Ser473) antibody (4060) and anti-p-mTOR antibody (Ser2448) (5536) were purchased from Cell signaling Technology (Beverly, USA). Rabbit anti-AKT antibody (ab32505) and anti-mTOR antibody (ab2732) were purchased from Abcam (Cambridge, UK). 4′, 6-diamidino-2-phenylindole (DAPI) (C1005) and insulin (P3376) were purchased from Beyotime Biotechnology (Haimen, China).
7
Exosome Quantification and Labeling
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The concentration of exosomes was determined using the FD™ BCA Protein Quantitative Kit (FDbio Science, Hangzhou, China). Exosomes were labeled with PKH26 using a membrane labeling kit (Sigma-Aldrich) according to the manufacturer's instruction. Briefly, PKH26 dye was diluted, added to the 10 μg of exosomes in 20-μL DPBS, and then incubated for 5 min after mixing by gentle pipetting. The excess dye was bound with 100 μL of 10% exosome-depleted FBS in RPMI 1640 medium. The exosomes were then diluted to 12 mL with DPBS and pelleted by ultracentrifugation at 100,000 × g at 4°C for 1 h 10 min. The pellet was resuspended in 50 μL DPBS. The imDCs were incubated with 5 µg/mL of PKH26-labeled exosomes for 12 h and stained with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO; C1993S; Beyotime, Guangzhou, China) and 4',6-diamidino-2-phenylindole (DAPI, C1005; Beyotime) before being observed with a Benchtop High-Content Analysis System (CQ1; Yokogawa, Musashino, Japan).
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