Three samples at PN5 were included, and at least three slices with good morphology from each sample were selected for immunofluorescence analysis. The procedures before primary antibody incubation were the same as the methods of immunohistochemistry mentioned above. The slices were incubated overnight (15 h) with diluted KLF6 mouse monoclonal antibodies (1:50, Santa Cruz, # sc-7158) and then treated with FITC conjugated goat anti-mouse IgG (Boster, #BA1101). Subsequently, the slices were treated with goat serum, following which the slices were incubated overnight (15 h) with diluted P21 rabbit polyclonal antibody (1:100, Proteintech, # 28248-1-AP). Wash four times with PBS. The slices were treated with Cy3 conjugated goat anti-rabbit IgG (Boster, #BA1032) in an immunohistochemical wet box. All the slices were counterstained with DAPI dye (Beyotime, #C1006)) for 5 min away from light. A Leica SP8 confocal laser scanning microscope was used to observe the fluorescence intensity. All slices were prepared under the same conditions and time to ensure homogeneity between samples. Images were taken under identical gain and offset values, with the same intensity of excitation wavelength and exposure time.
Dapi dye
Manufactured by Beyotime
Sourced in China
DAPI (4',6-Diamidino-2-Phenylindole) is a fluorescent dye commonly used in biological research. It binds strongly to DNA, emitting a blue fluorescence when excited by ultraviolet light. DAPI is a popular stain for visualizing and quantifying DNA in various applications, such as cell nuclei and chromosome counting.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dio dye, by Beyotime (2 mentions)
Cy3 conjugated goat anti rabbit igg, by Boster Bio (1 mentions)
Sp8 laser scanning confocal microscope, by Leica (1 mentions)
P21 rabbit polyclonal antibody, by Proteintech (1 mentions)
Fitc conjugated goat anti mouse igg, by Boster Bio (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Facscalibur, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apoptosis detection kit apc, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Beyotime (1 mentions)
Mf20 antibody, by Developmental Studies Hybridoma Bank (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Dhe probe, by Beyotime (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Dcfh da, by Beyotime (1 mentions)
15 protocols using dapi dye
1
Immunofluorescence Analysis of KLF6 and P21
2
Isolation and Purification of Torularhodin from Sporidiobolus pararoseus
Check if the same lab product or an alternative is used in the 5 most similar protocols
Sporidiobolus pararoseus JD-2 was obtained from the School of Biotechnology of Jiangnan University (China). Torularhodin was isolated and purified from the S. pararoseus extract according to a previously published method [25 (link)]. Its purity was greater than 95%, as determined by high-performance liquid chromatography with UV detection at 450 nm. Torularhodin was stored at -80°C; it was first dissolved in dimethyl sulfoxide (DMSO) and then in Dulbecco's modified Eagle's medium (DMEM) before use.
DMEM and fetal bovine serum (FBS) were from Gibco BRL (Grand Island, NY, USA). The DAPI dye and Cell Counting Kit-8 (CCK-8) were from Beyotime Biotechnology Co. Ltd. (Shanghai, China). The TRIzol Reagent was from Ambion (Kusatsu, Japan).
+ Expand
Sporidiobolus pararoseus JD-2 was obtained from the School of Biotechnology of Jiangnan University (China). Torularhodin was isolated and purified from the S. pararoseus extract according to a previously published method [25 (link)]. Its purity was greater than 95%, as determined by high-performance liquid chromatography with UV detection at 450 nm. Torularhodin was stored at -80°C; it was first dissolved in dimethyl sulfoxide (DMSO) and then in Dulbecco's modified Eagle's medium (DMEM) before use.
DMEM and fetal bovine serum (FBS) were from Gibco BRL (Grand Island, NY, USA). The DAPI dye and Cell Counting Kit-8 (CCK-8) were from Beyotime Biotechnology Co. Ltd. (Shanghai, China). The TRIzol Reagent was from Ambion (Kusatsu, Japan).
3
Apoptosis Quantification by FACS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected and detected by routine DAPI (4′,6‐diamidino‐2‐phenylindole) staining using a commercial DAPI dye (Beyotime, Shanghai, China) and Annexin V Apoptosis Detection Kit APC (eBioscience, San Diego, CA, USA) according to the manufacturers’ instructions and analysed by FACS Calibur. Briefly, 1 × 106 cells were washed and resuspended in 1× binding buffer. Fluorochrome‐conjugated Annexin V was added to the cell suspension and incubated at room temperature for 10 minutes. Cells were washed with 1× binding buffer. Propidium iodide (50 μg/mL) was added before flow cytometry analysis.
4
Immunofluorescence Staining of Sperm
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sperm slides were permeabilized and blocked with 5% goat serum (Beyotime, China) for 45 min. Primary antibodies were treated overnight at 4 °C. After three washes with PBS, Alexa Fluor 484- or 555-labeled donkey anti-rabbit IgG (Beyotime, China) was incubated for 45 min. The nuclei were counterstained with DAPI dye (Beyotime, China). The antibodies used in immunofluorescence staining are provided in Figure 7—source data 1 .
5
Myogenic Differentiation of P19 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence assay was performed in P19 cells with 1.0% DMSO treatment at 10 days. P19 cells were washed with PBS (Gibco) and fixed with 4% paraformaldehyde (Beyotime) for 15 min. Then, cells were permeated with 0.5% Triton X‐100 (Beyotime) for 20 min. The myosin heavy chain‐specific MF20 antibody (1:50, Developmental Studies Hybridoma Bank) was incubated for 2 h and the goat anti‐mouse secondary antibody labelled with red fluorescent was incubated for 1 h. Finally, the nucleus was labelled with DAPI dye (Beyotime). MF20 positive cells were observed by a fluorescence microscope (Nikon).
6
Virus Binding Assay in A549 and CEF Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 and CEF cells in 96-well-plates were incubated with 64 HA units of wild type (WT) or mutant viruses at 4 °C for 1 h. Then, cells were washed thrice in cold PBS and fixed for 1 h in 4% paraformaldehyde. The washing step with cold PBS was repeated thrice to prepare cells for the incubation with the corresponding chicken antiserum at a 1:500 dilution (1 h at 4 °C). The washing step with cold PBS was again repeated thrice, and cells were incubated for 1 h at 4 °C before adding a 1:1000 dilution of the secondary antibody (anti-chicken IgG conjugated with FITC; Life Technologies, USA). Cells were again washed thrice with cold PBS, and the nuclei were stained with DAPI dye (Beyotime Biotechnology, China) for 10 min at 37 °C, and observed under a fluorescence microscope (Nikon). The preparation of tissue sections has been described detailedly in our previous research article (Liu et al., 2022 (link)). The following steps were similar with the procedure described in virus binding to cells.
7
Cytosolic and Mitochondrial ROS Assays in Astrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cytosolic ROS detection, treated primary astrocytes were loaded with 10 μM DCFH-DA (Beyotime, S0033S) at 37 °C for 30 min. Cells were then washed with PBS three times before scanned by a microplate reader. For mitochondrial ROS detection in astrocytes, cells were loaded with 5 μM MitoSOX™ Red (Invitrogen, M36008) for 10 min and 200 nM Mito-Tracker Green (Beyotime, C1048) at 37 °C for 30 min. To visualize ROS production in the brain tissues, the slices were stained with 10 μM DHE probe (S0063) and DAPI dye (Beyotime, C1006). Images were viewed with a confocal scanning microscope.
8
EEC Uptake of DiO-Labeled AMEVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The uptake of AMEVs by EECs was performed as described previously.18 (link) Briefly, AMEV pellets (30 μL, approximately 8.04 × 1010±1.128 × 1010 particles) were incubated with a DiO dye (Beyotime Biotechnology, China) solution (10 μM) for 30 min at 37 °C. After washing twice with PBS, pellets were centrifuged at 100,000×g for 90 min, and DiO-AMEVs were resuspended in 100 μL of PBS. EECs (1 × 106/mL) were incubated with DAPI dye (Beyotime Biotechnology, China) (10 μg/mL) in a 15 mm glass bottom cell culture dish (NEST, Hong Kong, China) at 37 °C for 24 h, then washed three times with PBS and treated with DMEM containing 10 μL of DiO-AMEVs for 0 h, 48 h, and 72 h. Images were obtained using laser scanning confocal microscopy (Nikon Microsystems, Japan). Digital images were recorded and analyzed using NIS 4.2 Viewer (Nikon Microsystems, Japan).
9
Mechanism of BDE-47-induced Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BDE-47 (≥99.9%) was obtained from CSNpharm (Chicago, IL, USA). ISL (≥98%, HPLC) and LCB (≥98%, HPLC) were obtained from Yuanye (Shanghai, China). DCFH-DA and DMSO were obtained from Solarbio (Beijing, China). An apoptosis kit was obtained from Multi science (Hangzhou, China). A DAPI dye and neutral red staining solution were obtained from Beyotime Biotechnology (Shanghai, China). ELISA kits were obtained from Mlbio (Shanghai, China) (TNF-α Cat. No. ml002095; IL-6 Cat. No. ml063159; IL-1β Cat. No. ml301814). CAT, SOD, and GSH kits were obtained from Solarbio (Beijing, China). β-Actin, Nrf2, Keap1, HO-1, NQO1, IKBKB, IκB-Alpha, Bax, Bcl-2 antibodies, and the ECL luminescent reagent were obtained from Proteintech (Wuhan, China) (β-Actin Cat. No. 66009-1-lg; Nrf2 Cat. No. 16396-1-AP; Keap1 Cat. No. 60027-1-lg; HO-1 Cat. No. 66743-1-lg; NQO1 Cat. No. 67240-1-lg; IKBKB Cat. No. 15649-1-AP; IκB-Alpha Cat. No. 10268-1-AP; Bax Cat. No. 50599-2-lg; Bcl-2 Cat. No. 26593-1-AP). Caspase-3 antibody was obtained from BBI (Shanghai, China) (Cat. No. D16009). NF-κB and p-NF-κB antibodies were obtained from Abcam (Cambridge, UK) (NF-κB Cat. No. ab76302; p-NF-κB Cat. No. ab32536). P-Nrf2 antibody was obtained from Abclonal (Wuhan, China) (Cat. No. AP1133). Real-time fluorescent quantitative reagents were obtained from TransGen Biotech (Beijing, China).
10
BDE-47 Induced Apoptosis and Cytokine Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BDE-47 was purchased from AccuStandard (New Haven, CT, USA). DCFH2-DA, thiazole blue, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). The Annexin V-FITC/PI apoptosis kit was purchased from BD Bioscience (Franklin Lake, NJ, USA). Arg-1 and iNOS antibodies were purchased from eBioscience (San Diego, CA, USA). E. coli (strain K-12) was purchased from Invitrogen (Carlsbad, CA, USA). The IL-10 enzyme-linked immunosorbent assay (ELISA) kit was purchased from MultiSciences (Hangzhou, China). Cytochrome C antibodies, IL-6, IL-1β, TNF-α ELISA kits, DAPI dye, rhodamine 123, and Caspase-3 and Caspase-9 activity assay kits were purchased from Beyotime Biotechnology (Shanghai, China). BAX, Bcl-2, and TNF-α antibodies were purchased from WanLeiBio Co., Ltd. (Shenyang, China). ECL luminescent reagent was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Real-time fluorescent quantitative reagents were purchased from Takara Biomedical Technology Co., Ltd. (Beijing, China). N-acetylcysteine (NAC) and L-buthionine-(S,R)-sulfoximine (BSO) were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!