Cells were fixed in 4% paraformaldehyde for 15 min and incubated with 0.1% Triton X-100 (Beyotime, China) for 30 min. Additionally, tumor tissues were fixed in 4% paraformaldehyde, embedded with paraffin and cut into 5-µm sections. Then, the sections were incubated with goat serum to block nonspecific binding. The sections were subsequently incubated with anti-Ki67 antibody (1:50, Proteintech, China) or anti-Cytochrome C antibody (1:100, proteintech, China) overnight at 4 °C. After washing thrice with PBS, the sections were incubated with Cy3 goat anti-rabbit IgG (1:200, Beyotime, China) and counterstained with DAPI (Biosharp, China). The results were analyzed under a fluorescence microscope.
Cy3 goat anti rabbit igg
Manufactured by Beyotime
Sourced in China
Cy3 goat anti-rabbit IgG is a secondary antibody conjugated with the Cy3 fluorescent dye. It is designed to bind to rabbit primary antibodies, allowing for the detection and visualization of target proteins in various immunological techniques, such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342 dye, by Beyotime (2 mentions)
Tcs sp8, by Leica (2 mentions)
Anti ki67 antibody, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Biosharp (1 mentions)
Anti cytochrome c antibody, by Proteintech (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Fitc goat anti mouse igg, by Beyotime (1 mentions)
Lsm 700 confocal microscope system, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Rabbit anti h2a x antibody, by Cell Signaling Technology (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Eclipse te2000, by Nikon (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Normal goat serum (ngs), by Boster Bio (1 mentions)
Albumin bovine 5, by Biosharp (1 mentions)
Rabbit anti mpo, by Proteintech (1 mentions)
8 protocols using cy3 goat anti rabbit igg
1
Immunofluorescence Analysis of Ki67 and Cytochrome C
2
Immunofluorescence Analysis of JWA and HER2
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BT474 and SKBR3 cells were induced with JAC1 or vehicle (DMSO) for 24 h. Subsequently, the cells were fixed with methanol for 30 min, washed with PBST, and 10% normal goat serum for 1 h aiming to block non-specific signals. The cells were incubated with anti-JWA antibody (1:200) and anti-HER2 (1:250) overnight at 4 °C. After washing with PBST, the FITC goat anti-mouse IgG and CY3 goat anti-rabbit IgG (1:100, Beyotime, Jiangsu, China) were used to incubate with cells for 2 h. After washing three times with PBST, the cells were stained with DAPI (Beyotime, Jiangsu, China) for 20 min. The confocal images of the cells were captured using Zeiss AIM software on a Zeiss LSM 700 confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany).
3
Immunofluorescence Assay for NF-κB Activation
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BMDMs were plated in 24-well plates. After different stimulations, cells were washed thrice with cold PBS and fixed in 4% (w/v) paraformaldehyde for 20 min and then washed with PBS again. Next, cells were incubated in buffered normal goat serum to prevent nonspecific binding of antibodies for 1 h at room temperature. They then were incubated overnight with antibody against NFκB-65 (1 : 200, Cat# 8242S) purchased from Cell Signaling Technology (Massachusetts, USA), followed by incubation with Cy3 goat anti-rabbit IgG (1 : 500; Beyotime, Cat# A0516) for 1 h at 37°C. Thereafter, cells were washed in PBS. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 μM (Sigma, St. Louis, USA, Cat# D8417). Photomicrographs were taken with a Leica DMI3000B camera (Leica, Germany).
4
Inducible Cre-mediated Gene Manipulation
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Ad293 cells seeded overnight in 96-wells were transfected with pCMV-CreER, pCMV-mTEVp, pCMV-mCreER, or pCMV-mCreER/pCMV-mTEVp. Rapamycin and/or tamoxifen were added at 24 h after transfection. Cells were then fixed at 0 h, 24 h, or 48 h after induction with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.2% Triton X-100 for 10 min. Cells were incubated with the rabbit anti-H2A.X antibody (1:400, Cell Signaling Technology) or rabbit anti-Cre antibody (1:400, Cell Signaling Technology) for 2 h, and Cy3 goat anti-rabbit IgG (1:200, Beyotime, Shanghai, China) for 1 h, and finally with Hoechst 33,342 (10 μg/mL) (Beyotime, Shanghai, China) for 20 min. Three PBS washings were performed between each step. Images were taken under a fluorescence microscope equipped with a CCD camera (Nikon, Eclipse TE 2000, Tokyo, Japan).
5
Vitamin D Metabolism and Iron Homeostasis
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Iron Dextran (Yuanye Biology, S51662); VD3 (Beijing Solarbio, V8070); Tissue Iron Determination Kit (Nanjing Jiancheng, A039-2-1); hemoglobin (Hb) Kit (Nanjing Jiancheng, C021-1-1); red blood cell (RBC) diluent (Leagene Biotechnology, DA0150); 25-(OH)D3 kit (Jianglai Biological, JL20734); 1,25-(OH)D3 kit (Jianglai Biological, JL27246); transferrin (TF) (Jianglai Biotechnology, JL-31079); transferrin receptor (Trf) (Jianglai Biotechnology, JL-30566); serum iron (SI) (Nanjing Jiancheng, A039-1-1); iron-binding capacity (TIBC) (Nanjing Jiancheng, A040-1-1); CYP2R1 primary antibody (ABclonal, A10470); CYP27A1 primary antibody (ABclonal, A1982); CYP27B1 primary antibody (ABclonal, A1716); CYP24A1 primary antibody (ABclonal, A1805); Cy3-goat anti-rabbit IgG (Beyotime, A0516). Microplate reader (Bio-Rad, USA); DAPI (Aladdin, D106471); Paraffin embedding machine (Wuhan Junjie Electronics Co, Ltd, JT-12S); Microtome (Lycra Microsystems Co, Ltd, RM2016); Electron microscope (Nikon Instruments Co, Ltd, Eclipse Ci-L); PCR instrument (Hangzhou Bioet Company, Line Gene9640PCR).
6
Orai1 and NFκB Signaling in NEFA-treated BRL-3A Cells
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BRL-3A cells treated with 1.2 mM NEFAs and siOrai1 were cultured on the bottom well (Cellvis, Hangzhou, China) for 24 h and then fixed with 4% paraformaldehyde for 30 min at room temperature. BRL-3A cells were incubated with 3% Albumin Bovine V (Biosharp, Hefei, China), 5% normal goat serum (Boster, Wuhan, China), and 0.5% Triton in PBS (Biofroxx, Guangzhou, China) for 30 min at room temperature for blocking unspecific bindings. Then, rabbit anti-Orai1 and mice anti-NFκB p65 were incubated in cells overnight at 4°C in a humidified chamber. The cells were incubated with Cy3 goat anti-rabbit IgG (1:500, Beyotime) and goat anti-mice IgG-FITC (Santa Cruz Biotechnology) for 1 h at room temperature, respectively, after rinsing four times with PBS. The nuclei were stained with Hoechst 33342 dye (Beyotime) for 30 min at room temperature. Fluorescence and images were obtained with a confocal laser-scanning microscope (Leica TCS SP8).
7
Immunofluorescence Assay for Cell Markers
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Cells were seeded in 6-well plates and cultured overnight. Then, the cells were fixed in 3.5% paraformaldehyde and permeabilized in a solution of 0.1% bovine serum albumin (BSA) and 0.5% Triton X-100 at room temperature. After the blocking solution was washed out, the cells were incubated with primary antibodies against SPOCK1, E-cadherin, or vimentin for 60 min at 37°C and then washed with 0.1% BSA twice. After 60 min of incubation at 37°C with Cy3 Goat Anti-Rabbit IgG (Beyotime, Shanghai, China) and then washing with 0.1% BSA, the cells were counterstained with DAPI and imaged under a fluorescence microscope. The experiments were performed in triplicate.
8
Quantifying NET, MPO, and NE Formation in Neutrophils
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To assess NET formation, neutrophils combined in round coverslips (from 24-well plates) from different groups were fixed with 4% paraformaldehyde for 15 min at 4°C. After washing steps, the nuclei were stained with Hoechst 33342 dye (Beyotime) for 8 min at room temperature. Images were obtained with a confocal laser-scanning microscope (Leica TCS SP8; Leica) with 40×/1.3 numerical aperture differential interference contrast and analyzed with the instrument's software. The percentage of NET with all neutrophils visualized was calculated to assess NET formation.
To assess MPO and NE formation, neutrophils combined in round coverslips (from 24-well plates) from control and SCH cows were fixed with 4% paraformaldehyde for 30 min at room temperature and nonspecific binding sites were blocked with 3% Albumin Bovine V (Biosharp), 5% normal goat serum (Boster), and 0.5% Triton (BioFROXX) in PBS (Biosharp) for 30 min at room temperature. Cells were then incubated with rabbit anti-MPO (1:500, Proteintech)-NE (1:500, Biorbyt) at 4°C in a humidified chamber overnight. Cells were rinsed 4 times with PBS and incubated with Cy3 goat anti-rabbit IgG (1:1,000, Beyotime) for 1 h at room temperature. After 4 washing steps, the nuclei were stained with Hoechst 33342 dye (Beyotime). Images were obtained with a confocal laser-scanning microscope (Leica TCS SP8; Leica).
To assess MPO and NE formation, neutrophils combined in round coverslips (from 24-well plates) from control and SCH cows were fixed with 4% paraformaldehyde for 30 min at room temperature and nonspecific binding sites were blocked with 3% Albumin Bovine V (Biosharp), 5% normal goat serum (Boster), and 0.5% Triton (BioFROXX) in PBS (Biosharp) for 30 min at room temperature. Cells were then incubated with rabbit anti-MPO (1:500, Proteintech)-NE (1:500, Biorbyt) at 4°C in a humidified chamber overnight. Cells were rinsed 4 times with PBS and incubated with Cy3 goat anti-rabbit IgG (1:1,000, Beyotime) for 1 h at room temperature. After 4 washing steps, the nuclei were stained with Hoechst 33342 dye (Beyotime). Images were obtained with a confocal laser-scanning microscope (Leica TCS SP8; Leica).
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