Prior to immunofluorescence staining, monocytes were allowed to adhere to the glass bottom of confocal dishes for 2 hours. Cells were then washed 3 times (5 min each) with PBS (Phosphate Buffer Saline), fixed in 4% paraformaldehyde at room temperature for 15 min and then washed again in PBS 3 times (5 min each). Cells were blocked in 5% bovine serum albumin in PBS-T (PBS +0.2% Triton X-100) for 1 hour at room temperature. Primary antibodies (CD163, ab87099, Abcam and EpCAM, ab20160, Abcam) were diluted to 1:40 in blocking solution. 200uL of the primary antibody solutions were added to each dish, and cells were incubated overnight at 4°C. After incubation, cells were washed 3 times (5 min each) with PBS. All subsequent steps were performed in the dark. 200μl of secondary antibodies (TRITC goat anti-mouse IgG, ZF-0313, FITC goat anti-rabbit IgG, ZF-0313, 1:50 dilution in blocking solution) was added to each dish, and incubated for 60 min at room temperature. After incubation, excess secondary antibodies were removed and 200μl of DAPI (C0065, Solarbio) was added to each dish and incubated for 10 min at room temperature in the dark. Cells were then washed 3 times (5 min each) with PBS and observed under a confocal microscope (Leica).
C0065
Manufactured by Solarbio
Sourced in China, United States, Germany
C0065 is a laboratory equipment used for protein separation, purification, and analysis. It operates based on the principle of column chromatography. The core function of this product is to facilitate the separation and purification of proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Solarbio (9 mentions)
Axio observer a1, by Zeiss (6 mentions)
Ab150113, by Abcam (5 mentions)
P1110, by Solarbio (5 mentions)
T8200, by Solarbio (4 mentions)
Ab150080, by Abcam (4 mentions)
Confocal microscope, by Leica (3 mentions)
Zf 0511, by ZSGB-BIO (2 mentions)
Ab182981, by Abcam (2 mentions)
Las af lite software, by Leica (2 mentions)
Sa00013 4, by Proteintech (2 mentions)
M1000 pro, by Tecan (2 mentions)
A0521, by Beyotime (2 mentions)
A11037, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Ab20160, by Abcam (1 mentions)
Ab87099, by Abcam (1 mentions)
Ab183612, by Abcam (1 mentions)
Mab348, by Merck Group (1 mentions)
51 protocols using c0065
1
Monocyte Immunofluorescence Staining Protocol
2
Imaging Hippocampal Amyloid Pathology
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The hippocampal sections were isolated and perfused with PBS and 0.2% Triton X-100 and then fixed in 4% paraformaldehyde for 24 h. The left brain sections were rapidly frozen under -50°C, followed by transversely cutting the tissue samples into 5 μm slices. The slices were then incubated at 4°C with anti-APP (1 : 200, MAB348, Millipore, USA) and BACE1 (1 : 300, ab183612, Abcam, USA) antibodies. After permeabilization and blocking overnight, appropriate secondary antibodies (ab150113, ab150115, Abcam, USA) were used at a dilution of 1 : 200. After washing three times with PBS, the sections were incubated with DAPI (C0065, Solarbio, China) for 10 min, followed by live imaging. The hippocampal images were captured and obtained with a confocal laser microscope (FV1000, Olympus, Japan) at 1000x magnification.
3
Immunofluorescence Staining of CRC DLD1 Cells
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The CRC DLD1 cells were evenly seeded into the confocal dishes. When the cells in the dishes grew to an appropriate density followed by washing with PBS. Then, the cells were fixed using 4% paraformaldehyde at 4°C for the overnight storage, and after permeabilization with 0.1% TritonX−100/PBS, 5% BSA/PBS was used to block for 1 h. Then, the dishes were incubated with mouse anti-α-tubulin antibody (1:100, Proteintech Group) overnight at 4°C. Samples were stored at room temperature for 0.5 h, next washed three times in PBS and incubated Alexa Fluor 594 conjugated secondary antibody (1:100, ZF−0513, ZSGB-BIO, Beijing, China), and then kept in dark place at room temperature for 1 h. Nuclear stained with DAPI for 5 min (C0065, Solarbio, Shenzhen, China). Finally, the samples were imaged by Olympus confocal microscope. In addition, permeabilization and blocking were omitted when staining with Phalloidin (1:100, Cytoskeleton, Shanghai, China). It can be directly dyed in dark at room temperature for 1 h, and then re-stained with DAPI to take photos.
4
Confocal Microscopy of NPC Cells
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NPC cells were cultured in a six-well plate with glass slides. The cells were fixed with 4% Paraformaldehyde Fix Solution for 1h after adherence. After washing with PBS, the non-specific binding sites were sealed with goat serum at room temperature for 30 min. After absorbing goat serum with filter paper, the cells were incubated with primary antibodies at 4 °C overnight, washed with PBS for 3 times at the next day, and incubated with the secondary antibody at room temperature in darkness for 1 h, washed with PBS, stained with DAPI (Solarbio, C0065) for 3 min, and again washed with PBS. The anti-fluorescence quenching sealed tablet was then applied (Servicebio, G1401). Imaging was performed with a confocal laser microscope (Leica, Germany).
5
TFEB Nuclear Translocation Assay
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HepG2 cells cultured on glass dishes were pre-treated with Nit or Tiz (10 μmol/L) in the absence or presence of compound C (CC, 10 μmol/L, HY-13418A, MedChemExpress, USA) for 24 h. After being washed with PBS for 3 times, cells were subsequently fixed with 4% paraformaldehyde for 15 min, penetrated in PBS containing 0.4% Triton X100 for 1 h and blocked with goat serum (AR0009, Boster Biotech Technology, USA) for 30 min at room temperature. For monitoring nuclear translocation of TFEB, cells were then incubated with TFEB antibody (dil:1:200, A303-673A, Bethyl Laboratories Inc., USA) overnight at 4 °C, followed by incubation with the secondary antibody conjugated to Alexa Fluor 594 (dil:1:400, A-11037, Invitrogen) for 1 h, and the nuclei were counterstained with DAPI (10 μg/mL, C0065, Solarbio, Beijing, China) for 15 min. Fluorescence images were acquired by using a FV10i confocal microscope (OLYMPUS, Japan).
6
Immunofluorescent Staining of EPCs
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Immunofluorescent staining for CD34, CD133, VEGFR2, and β-catenin was conducted using EPCs after two passages. Briefly, these cells were plated in 12-well plates (1× 104/well) until 80% confluent, at which time they were fixed for 15 min using 4% paraformaldehyde (PFA), then permeabilized with 0.1% Triton X-100 for 20 min, and blocked for 30 min using 10% goat serum. These cells were then incubated overnight with mouse monoclonal anti-CD34 (12,034,042,1:50; eBioscience, USA), goat polyclonal anti-VEGFR2 (ab10972, 1:500; Abcam), rat monoclonal anti-CD133 (12,133,182, 1:100; eBioscience, USA), and/or mouse monoclonal anti-Beta-Catenin (138,400, 1:500; ThermoFisher) at 4 °C. Samples were then washed, probed for 1 h with secondary antibodies at 37 °C, and Nuclei were labeled with DAPI (C0065, Solarbio, China). Samples were then combined with 100 μl of anti-fluorescence attenuating mount, after which they were evaluated with an Intelligent Full-Automatic Fluorescence Microscopy Imaging System (Invitrogen™ EVOS™ FL Auto 2, Thermo Scientific, USA).
7
Immunofluorescence Analysis of Neuronal Markers
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After the H2O2 exposure, VSC4.1 cells were rinsed with PBS, fixed with 4% paraformaldehyde (G1101, Servicebio, Wuhan, China), and permeabilized with 0.3% Triton X‐100 (T8200, Solarbio) for 15 min. Nonspecific binding was blocked with 5% BSA Blocking Buffer (SW3015, Solarbio). Next, VSC4.1 cells were incubated with primary antibodies MAP‐2 (ab32454, 1:50, Abcam), NF‐H (#2836, 1:100, Cell Signaling Technology), α‐tubulin (#3873, 1:1000, Cell Signaling Technology), and β‐tubulin (#2128, 1:100, Cell Signaling Technology) overnight at 4°C. Cells were then incubated with secondary antibodies Alexa Fluor 647 (ab150107, Abcam, UK) and Alexa Fluor 488 (#4412, Cell Signaling Technology) for 1 h at room temperature and counterstained with DAPI (C0065, Solarbio) to visualize the nuclei. Finally, the cells were imaged under a fluorescence microscope (Olympus BX63), and the fluorescence intensity was quantified by ImageJ software (National Institutes of Health).
8
Immunofluorescent Analysis of LC3B and NLRP6
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Briefly, all sections (5 μm) were routinely deparaffinized in xylene, and antigen was retrieved by means of a 0.01 M sodium citrate-hydrochloric acid buffer. Slides were washed, blocked in 5% normal goat serum for 30 min, and stained overnight at 4°C using primary antibodies, including mouse anti-LC3B antibody (1:2,000; sc-271625; Santa Cruz, Texas, USA) and rabbit anti-NLRP6 antibody (1:1,000; 144-61128-50; Raybiotech, Guangzhou, China), and secondary antibodies conjugated to goat anti-mouse Alexa Fluor 488 (1:400; ab150113; Abcam, Fremont, CA, USA) or goat anti-rabbit Alexa Fluor 594 (1:300; ab150080; Abcam, Fremont, CA, USA). The nucleus was stained by DAPI (4′,6-diamidino-2-phenylindole; C0065, Solarbio, Beijing, China) and washed three times with PBS. The sections were photographed with a Nikon Eclipse TE 2000S inverted microscope (Nikon Instruments, Inc., Melville, NY). The positively stained puncta were counted using Image-Pro Plus software (Media Cybernetics, USA).
9
Mitigating AsO2-Induced Mitochondrial Dysfunction
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According to the departmental protocols, the effect of SeNPs supplementation on mitochondrial dysfunctions in renal cells induced by in vitro NaAsO2 exposure was analyzed by assessing mitochondrial activity and Mitochondrial Membrane Potentials (ΔΨm) of renal cells by MitoTracker staining and JC-1 staining.8 (link)
For MitoTracker staining, HK2 cells after drug treatments were thoroughly washed with Dulbecco’s phosphate buffered saline solution (DPBS, 14190250, Thermo Fisher Scientific, Beijing, China) and incubated with 4% paraformaldehyde solution (PFA, P1110, Solarbio, Beijing, China) at room temperature for 15 min. Subsequently, HK2 cells from each group were incubated with 200 nM MitoTracker staining solution (C1049, Beyotime, Shanghai, China) at 37 °C for 30 min. After incubation of 10 μg/mL DAPI staining solution (C0065, Solarbio, Beijing, China), the MitoTracker staining density of each group was analyzed by Image J software.
For JC-1 staining, HK2 cells after PFA fixation were incubated with 10 μM JC-1 staining solution (C2006, Beyotime, Shanghai, Zhejiang, China) at 37 °C for 20 min, followed by further incubation of DAPI staining solution and subsequent analysis of staining intensity by Image J software.
For MitoTracker staining, HK2 cells after drug treatments were thoroughly washed with Dulbecco’s phosphate buffered saline solution (DPBS, 14190250, Thermo Fisher Scientific, Beijing, China) and incubated with 4% paraformaldehyde solution (PFA, P1110, Solarbio, Beijing, China) at room temperature for 15 min. Subsequently, HK2 cells from each group were incubated with 200 nM MitoTracker staining solution (C1049, Beyotime, Shanghai, China) at 37 °C for 30 min. After incubation of 10 μg/mL DAPI staining solution (C0065, Solarbio, Beijing, China), the MitoTracker staining density of each group was analyzed by Image J software.
For JC-1 staining, HK2 cells after PFA fixation were incubated with 10 μM JC-1 staining solution (C2006, Beyotime, Shanghai, Zhejiang, China) at 37 °C for 20 min, followed by further incubation of DAPI staining solution and subsequent analysis of staining intensity by Image J software.
10
Visualizing NF-κB Nuclear Translocation
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The nuclear translocation of NF-κB was visualized using laser scanning confocal microscopy (LSCM). After the above treatments, the coverslips were thoroughly washed with three 2-min wash cycles using phosphate-buffered saline (PBS). Subsequent steps were fixation with 4% paraformaldehyde for 20 min, permeabilization with 0.5% Triton X-100 for 5 min, and blocking with 1% BSA for 30 min, each followed by identical wash cycles. Following blocking, cells were incubated at 37°C with primary NF-κB p65 antibody (2 μg/mL, ab76302, Abcam, United Kingdom) for 2 h. A post-incubation wash cycle was performed before the cells were exposed to a goat anti-rabbit IgG H&L secondary antibody (2 μg/mL, ab205718, United Kingdom) for 1 h at 37°C in the dark. Another wash cycle preceded DAPI counterstaining (1 μg/mL, C0065, Solarbio, Beijing, China) for 5 min at room temperature. After a final wash cycle, cells were mounted with 50% glycerol and imaged using a laser scanning confocal microscope (LSM700, Carl Zeiss AG, Germany).
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