The experimental protocol involved seeding cells into 96‐well plates in triplicate for the CCK‐8 (Dojindo Laboratories), followed by measurement of cell viability in accordance with the manufacturer's instructions. Colony formation was assessed by seeding cells into 6‐well plates (500 cells/well) in triplicate and culturing for 10 days, after which the colonies were fixed, stained, and counted. For the EdU assay kit (RiboBio), cells were seeded into 24‐well plates (3 × 104 cells/well) in triplicate, cultured for 24 h, then incubated with 50 μM EdU for 2 h. Subsequent staining and visualization were performed.76 The EdU assay involves the addition of a penetrant to cell climbing slides that have been prepared and subsequently incubated in a rocker device for 10 min. Subsequently, the EdU staining reaction solution was added and incubated at room temperature for 30 min in the dark. After three washes with phosphate‐buffered saline (PBS), the slides were incubated for 10 min at room temperature with DAPI solution (Servicebio). The liquid was then discarded, and the slide was covered with a fade‐resistant mounting solution. Finally, the images were collected and examined using fluorescence microscopy.
Dapi solution
Manufactured by Wuhan Servicebio Technology
Sourced in China
DAPI solution is a fluorescent dye used for staining and visualizing DNA in biological samples. It binds to the minor groove of DNA, emitting a blue fluorescence when excited by ultraviolet (UV) light. DAPI solution is a common tool used in various laboratory applications, such as fluorescence microscopy and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (2 mentions)
One step tunel apoptosis assay kit, by Beyotime (2 mentions)
Cy3 conjugated goat anti rabbit antibody, by Wuhan Servicebio Technology (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Edu assay kit, by RiboBio (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Airyscan, by Zeiss (1 mentions)
Cy3 conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Fitc conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Phosphate buffered saline (pbs), by Wuhan Servicebio Technology (1 mentions)
Cy3 labeled goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Trypsin, by Sartorius (1 mentions)
Keratinocyte sfm medium, by Thermo Fisher Scientific (1 mentions)
H e staining kit, by Abcam (1 mentions)
Anti akt, by Wuhan Servicebio Technology (1 mentions)
Dimethylsulfoxide dmso, by MedChemExpress (1 mentions)
Anti tnf α, by Wuhan Servicebio Technology (1 mentions)
Anti bax, by Wuhan Servicebio Technology (1 mentions)
Tunel assay kit, by Abcam (1 mentions)
19 protocols using dapi solution
1
Comprehensive Cell Viability and Proliferation Assays
2
Subcellular Localization of Viral Proteins
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Human embryonic kidney (HEK-293T) cells, African green monkey kidney epithelial (Vero) cells, porcine kidney (PK-15) cells, porcine testis (ST) cells, and porcine alveolar macrophage (3D4/21) cells were seeded in 6-well dishes and incubated at 37 °C in a 5% CO2 cell culture incubator to 70–90% confluency. Cells were transfected with the eukaryotic expression plasmids pEGFP-N1, pEGFP-Cap, pEGFP-NLS, pEGFP-Cap∆NLS, pEGFP-Cap∆NLS-A, and pEGFP-Cap∆NLS-B, respectively, using a lipid-based transfection method [43 (link)] (Hieff Trans® Liposomal Transfection Reagent, Yeasen, Shanghai, China) according to the manufacturer’s instructions.
Briefly, 10 μL of the lipid-based transfection reagent was added to 250 μL of Opti-MEMTM medium and incubated at room temperature for 5 min. An amount of 2–4 μg of plasmid DNA was added to 250 μL of Opti-MEMTM medium and mixed. Then, the reagents obtained above were mixed and incubated at room temperature for 20 min and added to a plate at 37 °C for 48 h. The cell culture supernatant was discarded, and the cells were fixed by adding 80% acetone and incubating at −80 °C for 2 h. After three washes with PBST, the cells were treated with DAPI solution (Servicebio, Wuhan, China) for 5 min in a dark room. Then, the cells were washed three times with PBST and examined using a laser confocal microscope (Zeiss LSM880 with Airyscan, Zeiss, Jena, Germany).
Briefly, 10 μL of the lipid-based transfection reagent was added to 250 μL of Opti-MEMTM medium and incubated at room temperature for 5 min. An amount of 2–4 μg of plasmid DNA was added to 250 μL of Opti-MEMTM medium and mixed. Then, the reagents obtained above were mixed and incubated at room temperature for 20 min and added to a plate at 37 °C for 48 h. The cell culture supernatant was discarded, and the cells were fixed by adding 80% acetone and incubating at −80 °C for 2 h. After three washes with PBST, the cells were treated with DAPI solution (Servicebio, Wuhan, China) for 5 min in a dark room. Then, the cells were washed three times with PBST and examined using a laser confocal microscope (Zeiss LSM880 with Airyscan, Zeiss, Jena, Germany).
3
Apoptosis Detection by TUNEL Assay
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To evaluate apoptosis, the TdT-mediated dUTP nick-end labeling (TUNEL) staining assay was used. It was performed using a one-step TUNEL apoptosis assay kit (Guge Biology, Wuhan, China) applied to the fixed midgut tissues. The cell nucleus was stained with the DAPI solution (Servicebio, Wuhan, China), at 37°C, for 10 min. The intestinal images were acquired under a fluorescence microscope (Leica DMI8, Wetzlar, Germany).
4
Immunofluorescence Staining of Type II Collagen, MMP13, and NF-κB
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For type II collagen and MMP13 immunofluorescence staining, NPCs were fixed in 4% paraformaldehyde for 15–20 min, then permeabilized using 0.1% v/v Triton X-100 for 5 min. Samples were incubated with primary antibodies against type II collagen (1:100 – 1:500, Solarbio, K009364P), MMP13 (1:400 – 1:1,600, Servicebio, GB11247), and NF-κB p65 (1:1,000, Cell Signaling Technology, Inc., Danvers, MA, United States, D14E12, #8242) diluted in 0.2% w/v BSA-TBS for 1 h. After washing with PBS, samples were incubated with DAPI solution (Servicebio, G1012) for visualization of the nuclei followed by incubation with FITC-conjugated goat anti-rabbit IgG (Servicebio, GB22303) and Cy3-conjugated goat anti-rabbit IgG (Servicebio, GB21301) for 30 min in the dark. Fluorescence detection was performed by fluorescence microscope (Olympus, Japan). Immunofluorescence quantification was performed using ImageJ, following the ImageJ User Guide.
5
Immunofluorescence Localization of JAK2 in Ovine Mammary Gland
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The localization of JAK2 in ovine mammary glands was detected using immunofluorescence staining, as described by Li et al. [44 (link)]. Briefly, slices of ovine mammary gland tissue were dewaxed using a dewaxing solution (Servicebio, Wuhan China) and then treated with antigen retrieval. Subsequently, the slices were washed with phosphate-buffered saline (PBS, Servicebio, Wuhan, China) and air-dried before being blocked with 5% BSA (Servicebio, Wuhan, China). After 30 min, the tissues were incubated with rabbit primary antibody against JAK2 (1: 100; Abmart, Shanghai, China) at 4 °C overnight. Cy3-labeled goat anti-Rabbit IgG (1: 300; Servicebio, Wuhan, China) was added and then incubated at room temperature for 1 h. The slices were washed three times with PBS before being stained with DAPI solution (Servicebio, Wuhan, China) and, then, incubated at room temperature for 10 min. Finally, the slides were sealed with anti-fluorescence quenching tablets (Servicebio, Wuhan, China) and, then, observed using a fluorescence microscope (Olympus, Tokyo, Japan).
6
BSDZG Modulates Inflammatory Pathways
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BSDZG was provided by Tianjiang Pharmaceutical Co., Ltd. (Jiangsu, China). Bicinchoninic acid (BCA) kit and, complete Freund’s adjuvant, and physiological saline were phrased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). H&E staining kit and TUNEL assay kit were bought from Abcam (Cambridge, UK). Dehydrocorydaline (DHC) and dimethyl sulfoxide (DMSO) were obtained from MedChemExpress (New Jersey, USA). Keratinocyte-SFM medium was phrased from Thermo Fisher Scientific Inc. (Massachusetts, USA). Fetal bovine serum (FBS) and trypsin were obtained from Biological Industries (Beit Haemek, Israel). Lipopolysaccharide (LPS) was brought from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Tumor necrosis factor-α (TNF-α) ELISA assay kit and interleukin-1β (IL-1β) ELISA assay kit were brought from Meibiao Biology (Nanjing, China). DAPI solution, anti-AKT, anti-phosphorylated AKT (p-AKT), anti-p38 MAPK, anti-phosphorylated p38 MAPK (p-p38 MAPK), anti-Bax, ani-Bcl-2, anti-NF-κB2, anti-TNF-α, anti-tumor necrosis factor receptor (TNFR), anti-GAPDH, and anti-vinculin were phrased from Servicebio Technology (Wuhan, China).
7
LPS-Induced Autophagy Regulation by HA
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Cells were seeded into 24-well plates containing the chamber slides and cultured overnight. Cells were treated with 10 μg/mL of LPS, followed by 40 μg/mL of different HA derivatives for 24 h. The cells growing on the chamber slides (cell-climbing films) were fixed with 4% paraformaldehyde and blocked with goat serum (Beyotime) for 1 h at 37 °C. Next, cells were incubated overnight at 4 °C with anti-LC3 primary antibody (Proteintech). Subsequently, cells were incubated with fluorescence-labeled goat anti-rabbit secondary antibody (MDL) for 1 h in the dark at 37 °C. Then, cells were stained with DAPI solution (Servicebio) for 10 min in the dark. Finally, cells were imaged under the Leica DM3000 fluorescent microscope.
8
Evaluating Chondroprotective Effects of High Molecular Weight Hyaluronic Acid
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LPS (Escherichia coli 055: B5) was obtained from Sigma-Aldrich (St. Louis, MO, USA). HMW-HA (0.8–1.5 × 106 Da, 1.8 × 106 Da) was obtain from Meilunbio (Dalian, China). CCK-8 and One Step TUNEL Apoptosis Assay Kit were purchased from Beyotime (Shanghai, China). DAPI solution and FITC conjugated Goat Anti-Rabbit IgG (H + L) was obtained from Servicebio (Wuhan, China). All primary antibodies used in this study were from Proteintech Group (Chicago, IL, USA) except the anti-β-actin (Medical Discovery Leader (MDL) Biotechnology, Beijing, China), anti-IL-1β (Abcam, Cambridge, UK) and anti-COL2A1 (Santa Cruz Biotechnology, Dallas, TX, USA). Bafilomycin A1 (Baf-A1) was purchased from Selleck (Houston, TX, USA). The reagents used for synthesis of o-HA derivatives were of analytical grade.
9
ROS Staining of Gill Tissue
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The refrigerant gill tissues were embedded by OCT (Sakura, USA), then sliced by freezing microtome (Thermo, CRYOSTAR NX50, US). The slice thickness was 8–10 μm. The frozen slides were restored to room temperature, then incubated with spontaneous fluorescence quenching reagent (Servicebio, China). ROS staining solution (Servicebio, Wuhan, China) was used to stain slides at 37℃ for 30 min in dark place, following washing three times with PBS (pH 7.4) and then incubating with DAPI solution (Servicebio, Wuhan, China) at room temperature for 10 min in dark place. Finally, the sections were observed by Fluorescent Microscopy (Pannoramic MIDI, 3DHISTECH, HUN).
10
Immunofluorescence Analysis of Urogenital Tissues
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Paraffin sections, providing a clear and comprehensive view of the urethra, hindgut, URS, and rectourethral fistulas, were chosen for staining. Subsequently, the sections underwent deparaffinization and antigen retrieval. Blocking was carried out with serum (ZSGB-BIO, Beijing, China) at 25 °C for 1 hour, followed by overnight incubation at 4 °C with primary antibodies. The primary antibodies used were as follows: Anti-Pcsk9 (1:100), anti-Hmgb2 (1:100), and anti-Sod1 (1:100), all sourced from Proteintech (Wuhan, China). After PBS washes, the sections were incubated at 25 °C in the dark for 1 hour with fluorescent secondary antibodies: goat anti-rabbit 488 (1:100; Proteintech). Following PBS washes, the sections were stained with DAPI solution (Servicebio Technology, Wuhan, China) for 10 minutes. Finally, the sections were mounted using an anti-fluorescence fading medium (Solarbio, Beijing, China), and images were captured using a laser-scanning confocal microscope (LSM 880, Zeiss, Oberkochen, Germany).
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