The cultured cells were fixed in 4% paraformaldehyde for 20 min at room temperature, subsequently washed with PBS and treated with 0.3% Triton X-100(Sigma) in PBS for 15 min. Then the samples were incubated with PBS containing 2% bovine serum albumin (BSA) at 37°C for 10 min. Incubation with the primary antibodies(mouse anti-c-kit, Santa Cruz, 1:200; Rabbit anti-vimentin, Abcam, 1:100; Rabbit anti-CD34, Bioss, 1:300; Rabbit anti-Nanog, Santa Cruz, 1:100; Rabbit anti-sca-1, Millipore,1:200) was performed at 4°C overnight. The samples were subsequently incubated FITC- or Cy3-conjugated secondary antibodies (Beyotime). The nuclei were counterstained with 1μg /ml DAPI (Roche). Negative controls were obtained by following all the same protocol but the primary antibodies. All experiments were performed in triplicate.
Cy3 conjugated secondary antibody
Manufactured by Beyotime
Sourced in China
Cy3-conjugated secondary antibody is a fluorescent-labeled antibody that binds to and detects primary antibodies. It is commonly used in immunofluorescence and other imaging techniques to visualize the location and distribution of target proteins or molecules within a sample.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Lsm t pmt confocal microscope, by Zeiss (3 mentions)
Fitc conjugated secondary antibody, by Beyotime (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Rabbit anti nanog, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti vimentin, by Abcam (1 mentions)
Rabbit anti sca 1, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Total internal reflection fluorescence microscope, by Nikon (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti von willebrand factor, by Abcam (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Anti iba1, by Abcam (1 mentions)
Ab62352, by Abcam (1 mentions)
Ultraview vox confocal microscope, by PerkinElmer (1 mentions)
Model ix83, by Olympus (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
25 protocols using cy3 conjugated secondary antibody
1
Immunofluorescence Staining of Cultured Cells
2
Immunofluorescence Analysis of EMT Markers
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At 48 h after RNA transfection, cells from different groups were seeded onto coverslips in 6-well dishes. After completely adhered to the coverslips, cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 1% (v/v) Triton X-100 for 10 min at room temperature (RT), and then blocked with 1% goat serum for 1 h at RT. Subsequently, the coverslips were incubated with primary antibodies (anti E-cadherin, Abcam; anti vimentin, Abcam) at 4℃ overnight. Next day, the coverslips were washed three times with PBS and incubated with Cy3- conjugated secondary antibodies (Beyotime) for 1 h at RT, then all the coverslips were counterstained with 4’-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Finally, photomicrographs were captured with a Nikon Total Internal Reflection Fluorescence microscope. The semi-quantitative analysis of immunofluorescence staining was conducted using Image Pro Plus software.
3
Immunofluorescence Staining of Brain Tissues
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Immunofluorescence staining was performed at 72 h after the operation. Briefly, the mice were anesthetized and perfused with 4% paraformaldehyde. Brain specimens were rapidly collected and postfixed in 4% paraformaldehyde for 1 day and a 30% sucrose solution for the following 3 days. Fifteen μm thick coronal sections were cut and treated with 0.3% Triton for 30 min. The brain sections were subsequently treated with 5% donkey serum for 1 h for antigen block and incubated with the primary antibodies overnight in a 4°C freezer, including anti-zonula occludens-1 (1 : 100, Invitrogen, Grand Island, NY), anti-von Willebrand factor (1 : 100 Abcam), anti-Iba1 (1 : 100, Abcam), and anti-MST1 (1 : 100, Invitrogen, Grand Island, NY), followed by FITC-conjugated secondary antibody (1 : 200, Beyotime Biotechnology, Shanghai, China) and cy3-conjugated secondary antibodies (1 : 200, Beyotime Biotechnology, Shanghai, China) for 1.5 h at room temperature. A confocal laser scanning microscope (Zeiss, Oberkochen, Germany) was used to detect the expression of fluorescent dyes.
4
Macrophage Identification in Liver Tissue
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Frozen sections of liver tissue were prepared immediately after sampling and then incubated with F4/80 (1:300, CST, USA) antibodies at 4℃ overnight. The sections were incubated with Cy3-conjugated secondary antibodies (1:300, Beyotime) and stained with DAPI (Beyotime). Finally, a ZEISS 780 laser confocal microscope was used to capture fluorescence images and analyze macrophage clearance by immunofluorescence staining.
5
Nrf2 Immunofluorescence Staining Protocol
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Cells were washed with PBS, fixed with 4 % formaldehyde and blocked with 1 % BSA/PBS for 1 h at room temperature. Then, cells were incubated with a primary antibody against Nrf2 (Abcam; ab62352) overnight at 4 °C and incubated with Cy3-conjugated secondary antibodies (Beyotime, Nantong, China) for 1 h at room temperature. DAPI (Sigma) was used to stain the nuclei and images were captured using an UltraViewVoX confocal microscope (PerkinElmer, Waltham, MA).
6
Immunohistochemical Analysis of Brain Tissue
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For immunohistochemical staining of brain tissues, frozen brain sections were deparaffinized and subjected to microwave heat-induced epitope retrieval. The sections were blocked with 10% normal goat serum for 30 min and incubated with primary antibodies against NSE (1:100, Abcam) or NF-200(1:100, Abcam) overnight at 4°C. The sections were then incubated with appropriate Cy3-conjugated secondary antibodies (1:200, Beyotime) for 60 min at room temperature. Images were obtained at a magnification of 400× from 5 random microscopic fields using a fluorescence microscope.
7
Immunofluorescence Analysis of Morphine-Treated Cells
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Cells were seeded into 12‐well plates covered with cell slides made of high‐quality glass (WHB‐12‐CS, WHB scientific, Shanghai, China) at a density of 2 × 104 cells/well and treated with morphine hydrochloride (MH) for 24 h. Next, cell slides were fixed with 4% paraformaldehyde (Servicebio, Wuhan, China) for 20 min and treated with 0.1% Triton X‐100 (P0096, Beyotime) for 10 min. Then, cell slides were blocked with 3% bovine serum albumin (Beyotime) in phosphate buffer (PBS; Corning, New York, USA) for 1 h at room temperature and incubated with primary antibody (MOR, 1:400, Cat# ab134054, Abcam; CK19, 1:400, Cat# ab52625, Abcam; N‐cadherin, 1:400, Cat# 13116, Cell Signaling Technologies; Vimentin, 1:400, Cat# 5741, Cell Signaling Technologies; Slug, 1:400, Cat# 9585, Cell Signaling Technologies) overnight at 4°C. Finally, slides were incubated for 1 h with Cy3‐conjugated secondary antibody (Beyotime), and then with DAPI (Vector, Newark, USA) for 10 min at room temperature. Representative fields were imaged with a laser confocal microscope (Model IX83, Olympus, Tokyo, Japan).
8
Immunofluorescence Analysis of B7-H3 Expression
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Cells were incubated in 24-well plates under standard cell culture conditions (5 × 103 cells per well). After 12 h, cells were blocked with 5% BSA for 15 min, stained with B7-H3 antibody (Abcam, ab227679) for 1 h, Cy3-conjugated secondary antibody (Beyotime, A0516) for 40 min and DAPI (Beyotime) in the dark. Images were captured on a fluorescence microscopy.
Tumor tissues from the T cell group mice were collected and immediately froze at −80°C. Sections were fixed in pre-chilled acetone-methanol (1:1) for 20 min at −20°C and then allowed to air-dry for 10 min before being blocked with 5% BSA for 30 min. Subsequently, sections were stained with B7-H3 antibody (Abcam, ab227679) for 1 h, FITC-conjugated secondary antibody (Beyotime, A0562) for 40 min and DAPI (Beyotime) in the dark. Images were captured on a fluorescence microscopy.
Tumor tissues from the T cell group mice were collected and immediately froze at −80°C. Sections were fixed in pre-chilled acetone-methanol (1:1) for 20 min at −20°C and then allowed to air-dry for 10 min before being blocked with 5% BSA for 30 min. Subsequently, sections were stained with B7-H3 antibody (Abcam, ab227679) for 1 h, FITC-conjugated secondary antibody (Beyotime, A0562) for 40 min and DAPI (Beyotime) in the dark. Images were captured on a fluorescence microscopy.
9
Immunofluorescence Staining of MRC-5 Cells
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Fixed MRC-5 cells were blocked with 10% goat serum (Beyotime, China) for 1 h at room temperature, incubated with primary antibody at 4 °C overnight, reacted with Cy3-conjugated secondary antibody (Beyotime, China) for 1 h at room temperature, dyed the nuclei with DAPI (Beyotime, China). Wash with PBST three times for 5 min between each step. The images were acquired by Fluoview 300 confocal laser scanning microscopy (Olympus, Tokyo, Japan).
10
Immunofluorescence Staining of Bovine Myoblasts
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Bovine myoblasts in 12-well plates were washed three times with PBS, fixed with 4% formaldehyde solution for 20 min, and permeabilised with 0.5% Triton X-100 in sterile water for 10 min. The cells were blocked with 5% BSA in PBS for 30 min and then incubated at 4°C with myosin antibody (Abcam, USA) overnight. After washing cells with PBS, the cells were incubated with Cy3-conjugated secondary antibody (Beyotime) diluted 1:200 in 1% BSA in the dark at room temperature for 2 h. DAPI staining was done simultaneously to show the position of the nuclei. All images were taken on an inverted fluorescence microscope (AMG EVOS).
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