The skin tissue sections were dewaxed and hydrated. After antigen retrieval treatment, anti-α-SMA antibodies (1:100 dilution, ab5694, Cambridge, UK), anti-TNF-α antibodies (1:100 dilution, GTX110520, San Antonio, TX, USA), or anti-vimentin antibodies (1:200 dilution, ab92547, Cambridge, UK) were added dropwise to the tissues and incubated overnight at 4 °C. Sheep anti-murine antibody (H + L) FITC (121,051, SeraCare, Milford, MA, USA) was then added at 37 °C for 1 h. Then, the tissues were stained with a dilution of 5 mg/mL 4′,6-diamidino-2′-phenylindole (DAPI; 14,285, Cayman Chemical Company, Ann Arbor, MI, USA) for 5 min. An Olympus DP80 microscope (Olympus, Hamburg, Germany) was used to observe the expression sites and levels of α-SMA, TNF-α, and vimentin. The obtained images were analyzed using cellSens Imaging Software v.1.8 (Olympus, Hamburg, Germany). Green fluorescence was taken to indicate α-SMA/TNF-α/vimentin expression.
4 6 diamidino 2 phenylindole dapi
Manufactured by Cayman Chemical
Sourced in United States
4′,6-diamidino-2′-phenylindole (DAPI) is a fluorescent dye that binds to the minor groove of DNA. It is commonly used in fluorescence microscopy and flow cytometry applications for the detection and quantification of DNA.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Thermo Fisher Scientific (2 mentions)
Dp80 microscope, by Olympus (1 mentions)
Antifade medium, by Beyotime (1 mentions)
Inverted fluorescent microscope, by Nikon (1 mentions)
Donkey anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
Goat anti mouse igg h l alexa fluor 568, by Abcam (1 mentions)
Rabbit anti cd34, by Abcam (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Adipogen (1 mentions)
Goat anti mouse igg h l alexa fluor 647 ab150115, by Abcam (1 mentions)
Imd 0354, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mouse anti pax6, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Proteintech (1 mentions)
Rabbit anti ctip2, by Abcam (1 mentions)
Rabbit anti tbr1, by Proteintech (1 mentions)
Rabbit anti pax6, by Proteintech (1 mentions)
Alexa fluor 555 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rat anti brdu, by Abcam (1 mentions)
6 protocols using 4 6 diamidino 2 phenylindole dapi
1
Immunofluorescence Staining for α-SMA, TNF-α, and Vimentin
2
Immunofluorescent Characterization of Cells
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Fresh cells were seeded at a suitable density on microscope slides, washed three
times with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized by
treating with 0.5% Triton X-100 for another 10 min. The cells were blocked by
treating with 3% bovine serum albumin (BBI, Shanghai, China) for 1 h. Rat
anti-vimentin (1:100; Cell Signaling Technologies, Danvers, MA, USA) and rabbit
anti-CD34 (1:200; Abcam, Cambridge, UK) antibodies were used. The cells were
treated with the antibodies overnight at 4°C. After washing three times with
PBS, the cells were treated with donkey anti-rabbit IgG (H+L) Alexa Fluor 488
(1:1000; Abcam, Cambridge, UK) or goat anti-mouse IgG (H+L) Alexa Fluor 568
(1:1000; Abcam, Cambridge, UK) at 37°C for 1 h, followed by treatment with
4’,6-diamidino-2-phenylindole (DAPI; Cayman Chemical, Ann Arbor, MI, USA). The
slides were fixed in an antifade medium (1:1000; Beyotime, Shanghai, China) and
imaged using an inverted fluorescent microscope (Nikon, Tokyo, Japan).
times with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized by
treating with 0.5% Triton X-100 for another 10 min. The cells were blocked by
treating with 3% bovine serum albumin (BBI, Shanghai, China) for 1 h. Rat
anti-vimentin (1:100; Cell Signaling Technologies, Danvers, MA, USA) and rabbit
anti-CD34 (1:200; Abcam, Cambridge, UK) antibodies were used. The cells were
treated with the antibodies overnight at 4°C. After washing three times with
PBS, the cells were treated with donkey anti-rabbit IgG (H+L) Alexa Fluor 488
(1:1000; Abcam, Cambridge, UK) or goat anti-mouse IgG (H+L) Alexa Fluor 568
(1:1000; Abcam, Cambridge, UK) at 37°C for 1 h, followed by treatment with
4’,6-diamidino-2-phenylindole (DAPI; Cayman Chemical, Ann Arbor, MI, USA). The
slides were fixed in an antifade medium (1:1000; Beyotime, Shanghai, China) and
imaged using an inverted fluorescent microscope (Nikon, Tokyo, Japan).
3
Dynorphin Peptide Characterization
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DYN 1–17, and a range of N-terminal fragments (DYN 1–6, DYN 1–7, DYN 1–9, DYN 1–10 and DYN 1–11) were supplied by Mimotopes Pty. Ltd, Australia. Purity was greater than 95% for all peptides. LPS from Escherichia coli, serotype O55:B5 was purchased from Enzo Life Sciences (Farmingdale, NY, USA), phorbol 12-myristate 13-acetate (PMA) was purchased from Adipogen (San Diego, CA, USA) and 4',6-diamidino-2-phenylindole (DAPI) was purchased from Cayman Chemical (MI, USA). Primary rabbit monoclonal anti-NF-κB/p65 [E379] antibody (ab32536), goat anti-rabbit IgG H&L (Alexa Fluor 555; ab150078) and goat anti-mouse IgG H&L (Alexa Fluor 647; ab150115) were provided by Abcam (Cambridge, MA, USA). The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent, dimethyl sulfoxide (DMSO), IMD-0354 and foetal bovine serum (FBS) were purchased from Sigma Aldrich (St Louis, MO, USA) whereas penicillin-streptomycin (Pen-Strep) solution and RPMI 1640 medium were supplied by Life Technologies (VIC, Australia). Primary mouse monoclonal KOP receptor antibody was obtained from Life Research Pty. Ltd., Australia.
4
Immunostaining Markers in Neurodevelopment
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Primary antibodies used in this study include: rabbit anti-Brn2 (Santa Cruz, 1:50), rabbit anti-Ctip2 (Abcam, 1:1000), rabbit anti-Tbr1 (Proteintech Group, 1:500), rat anti-BrdU (Abcam, 1:1000), rabbit anti-Ki67 (Abcam, 1:500), mouse anti-Pax6 (Cell Signaling Technology, 1:200), rabbit anti-Pax6 (Proteintech Group, 1:1000). Secondary antibodies used include: Alexa Fluor 555 donkey anti-rabbit IgG (Invitrogen, 1:300), Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, 1:300), Alexa Fluor 488 goat anti-mouse IgG (Proteintech Group, 1:300), Alexa Fluor 488 goat anti-rat IgG (Jackson Immuno Research, 1:300). Cell apoptosis was detected by TUNEL FITC Apoptosis Detection Kit (Vazyme, A111). Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Cayman Chemical).
5
Formulation and Characterization of PLGA Nanoparticles
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PLGA (50:50, Mw35000, Lack Siomaterials, USA); 4′, 6-diamidino-2-phenylindole (DAPI) (Cayman Chemical, Ann Arbor, MI, USA); 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiL) were purchased from Biotium (Fremont, CA, USA); phosphate-buffered saline (PBS), Roswell Park Memorial Institute medium (RPMI-1640), Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin solution (5 kU mL−1), fetal bovine serum (FBS), and trypsin were purchased from Life Technologies (Carlsbad, CA, USA); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl), (18:1 Liss RHod PE) and N-[6-[(7-nitro-2-2,3-benzoxadiazol-4-yl)amino]hexanoyl]-phytosphingosine, (C6-NBD phytosphingosine) were purchased from Avanti Polar Lipids (Birmingham, AL, USA).
4T1 with stable luciferase expression (4T1-luc cells), 4T1 breast cancer cells, and RAW264.7 macrophage cells were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). RAW264.7 cells were cultivated in DMEM media, whereas 4T1-luc and 4T1 cells were cultured in RPMI 1640 media supplemented with 10% FBS. All media contained antibiotics (100 U mL−1 penicillin and streptomycin) and 10% FBS.
4T1 with stable luciferase expression (4T1-luc cells), 4T1 breast cancer cells, and RAW264.7 macrophage cells were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). RAW264.7 cells were cultivated in DMEM media, whereas 4T1-luc and 4T1 cells were cultured in RPMI 1640 media supplemented with 10% FBS. All media contained antibiotics (100 U mL−1 penicillin and streptomycin) and 10% FBS.
6
Lipoic Acid Nanoparticle Delivery for Cancer Treatment
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Material used in this experiment were lipoic acid (Huayu Pharmaceutical Co., Ltd., Wuxi China); cysteine hydrochloride, sodium cholate, methanol and docetaxel (Sangon Biotech, Shanghai, China); PLGA (50:50, Mw35000, Lack Siomaterials, US); chlorpromazine hydrochloride and amiloride hydrochloride (Sigma-Aldrich, (St. Louis, MO, US); 4′,6-diamidino-2-phenylindole (DAPI) and Filipin III (Cayman Chemical, Michigan, US); 1,1-Dioctadecyl −3,3,30,30 – tetramethyl indotricarbocyanineiodide (DiR) (Biotium, CA, US); Apoptosis Assay Kit and Cell Cycle and Annexin V-FIT Kit (Becton Dickinson and Company, US); zeocin and puromycin (Invitrogen, Carlsbad, CA, US); RPMI-1640, foetalbovine serum (FBS), PBS and trypsin (Life Technologies, Grand Island, US).
Female nude mice aged 5 weeks and weighing about 16 g were purchased from the Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). All animals were raised at the Second Military Medical University (Shanghai, China).
All animal experiments were performed in accordance with the protocols evaluated and approved by the ethics committee of the Second Military Medical University.
Female nude mice aged 5 weeks and weighing about 16 g were purchased from the Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). All animals were raised at the Second Military Medical University (Shanghai, China).
All animal experiments were performed in accordance with the protocols evaluated and approved by the ethics committee of the Second Military Medical University.
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