Heart sections were deparaffinized and subjected to antigen retrieval in hot citric acid buffer (PH 6.0) for 20 min. After cooling, slides were blocked with 5% BSA in TBST for 1 h and incubated overnight with primary antibodies against ANGPTL4 (1:100, Cat# A13425, Abclonal) at 4°C overnight, followed by incubation with a goat anti-rabbit-Cy3 (1:200, Cat# GB21303, Servicebio) for 1 h at room temperature. Sections were washed with PBS three times and then counterstained with 4’,6-diamidino-2-phenylindole fluorescent dye (DAPI). While the AC16 cells were fixed with 4% paraformaldehyde for 10 min, then washed with PBS three times. The cells were blocked with normal goat serum and incubated with anti- ANGPTL4 (1:100, Cat# A13425, Abclonal) antibody at 4°C overnight. Cells were washed with PBS three times and then incubated with a goat anti-rabbit-Cy3 (1:200, Cat# GB21303, Servicebio) or anti-rabbit-FITC (1:200, Cat# GB22303, Servicebio) for 1 h. Cells were washed with PBS three times and then counterstained with DAPI. Digital images were observed under a microscope with SOPTOP ICX41 or OLYMPUS.
Cy3 goat anti rabbit
Manufactured by Wuhan Servicebio Technology
Sourced in China
CY3-goat anti-rabbit is a secondary antibody that is conjugated with the fluorescent dye Cyanine 3 (CY3). It is designed to detect and bind to primary antibodies raised in rabbit. The CY3 fluorescent label allows for visualization and detection of the target protein or antigen in various immunological techniques, such as immunofluorescence microscopy and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Anti ki67, by Wuhan Servicebio Technology (3 mentions)
A1 spectral confocal microscope, by Nikon (2 mentions)
Anti cd3, by Wuhan Servicebio Technology (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti gfap, by Wuhan Servicebio Technology (1 mentions)
Alexa fluor 488 goat anti rabbit, by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
Ckx53, by Olympus (1 mentions)
Gb21303, by Wuhan Servicebio Technology (1 mentions)
F4 80, by Wuhan Servicebio Technology (1 mentions)
Gb11027, by Wuhan Servicebio Technology (1 mentions)
Cd206, by Wuhan Servicebio Technology (1 mentions)
Eclipse e100, by Nikon (1 mentions)
Gb23303, by Wuhan Servicebio Technology (1 mentions)
Gb11059, by Wuhan Servicebio Technology (1 mentions)
Gb113109, by Wuhan Servicebio Technology (1 mentions)
Gb13014, by Wuhan Servicebio Technology (1 mentions)
Gb12068, by Wuhan Servicebio Technology (1 mentions)
Gb22302, by Wuhan Servicebio Technology (1 mentions)
Gb121141, by Wuhan Servicebio Technology (1 mentions)
6 protocols using cy3 goat anti rabbit
1
Immunodetection of ANGPTL4 in Heart Tissue and Cells
2
Immunostaining of Neural Stem Cells
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The SCs were fixed in 4% paraformaldehyde for 30 minutes at 4°C, blocked and incubated with monoclonal anti‐S100 (1:200),anti‐GFAP (1:200) and anti‐Ki67 (1:200, Servicebio, Wuhan, China) primary antibodies overnight at 4°C. The Alexa Fluor 488 goat anti‐rabbit (1:200, Servicebio) or Cy3 goat anti‐rabbit (1:200, Servicebio) was used as the secondary antibody, and DAPI (Servicebio) was utilized to detect the nucleus. The images were obtained using an inverted fluorescence microscope (CKX‐53, Olympus, Japan).
3
Immunofluorescence Staining of Mouse Lung Tumor Tissues
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Mouse lung tumor tissues were made into paraffin-embedded sections. For immunofluorescence staining, slides were fixed with 4% paraformaldehyde, permeabilized with 0.15% Triton X-100, blocked with 3% BSA for 30 min at room temperature (RT), and incubated with primary antibodies (F4/80, GB11027, Servicebio, 1:4000; CD24, DF8518, affinity, 1:100) in phosphate-buffered saline. Then, slides were incubated with secondary antibodies (Cy3-Goat anti-rabbit, GB21303, Servicebio, 1:300). For immunofluorescence double staining, slides were incubated with primary antibodies (F4/80, GB11027, Servicebio, 1:4000; CD86, DF6332, affinity, 1:200; CD206, GB13438, Servicebio, 1:500) in phosphate-buffered saline. Then, slides were incubated with secondary antibodies (HRP-Goat anti-rabbit, GB23303, Servicebio, 1:500; Cy3-Goat anti-rabbit, GB21303, Servicebio, 1:300) and FITC-Tyramide (G1222-50UL, servicebio). After staining, sections were placed under a microscope (Nikon Eclipse E100, Tokyo, Japan) for observation. Images were analyzed using Image-Pro Plus 6.0 software.
4
Immunohistochemical Analysis of Tumor Microenvironment
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After fixation in 4 % PFA, the tumor tissues and lymph nodes were dehydrated in a 20 % sucrose solution, embedded in paraffin, and subsequently cut into slices (5 μm). After incubation with 5 % BSA for 1 h, primary antibodies were added (anti-CD3, 1:500, GB13014; anti-CD4, 1:500, GB11064; anti-CD8, 1:2000, GB12068; anti-Foxp3, 1:500, GB13445; anti-CD68, 1:200, GB113109; anti-Ki67, 1:600, GB121141; anti-CD86, 1:300, GB13585; anti-CD11c, 1:300 GB11059, all from Servicebio, Wuhan, China) at 4 °C in a dark, wet box overnight. After extensive washing using FACS buffer, sections were incubated with secondary antibodies (FITC-goat anti-rat, 1:200, GB22302; CY3-goat anti-rabbit, 1:500, GB21301; Alexa Fluor 488-goat anti-mouse, 1:500, GB25301, all from Servicebio) for 1 h at 25 °C. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA). The stained sections were observed using a Nikon A1 spectral confocal microscope (Nikon, Tokyo, Japan).
5
Multiparameter Immunofluorescence Tissue Analysis
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TDLNs and other tissues of interest were harvested, and tissue sections of 8-μm thickness were prepared with a cryostat. After incubation with 5% BSA for 1 h, the sections were incubated with primary antibody (anti-CD3, 1:500, Servicebio, China, GB13014; anti-Foxp3, 1:500, Servicebio, China, GB13445; anti-LYVE1, 1:100, Abcam, USA, Ab14917; anti-Ki67, 1:800, Servicebio, China, GB111141) overnight at 4°C in a wet, dark box. After being washed three times, the sections were incubated with the fluorescence-labeled secondary antibodies (CY3-goat anti-rabbit, 1:300, Servicebio, China, GB21303; 488-goat anti-rabbit, 1:300, Servicebio, China, GB25303) for 1 h at room temperature. Cell nuclei were counterstained with Hoechst33342. The sections were washed with PBS and then observed with a Nikon A1 Spectral Confocal Microscope (Nikon, Japan).
6
Immunohistochemistry Analysis of Inflammatory Markers
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Immunohistochemistry was carried out by using S100A8/A9 (1:1000, Abcam, UK), Col1 antibody (1:1000, Proteintech, China), α-SMA (1:500, Proteintech, China), RAGE (1:200, Proteintech, China), p-JAK2(Tyr1007) (1:100, Absin, China), p-STAT3 (Tyr705) (1:100, Absin, China), CK18(1:1000, Proteintech, China), and vWF (1:1000, Proteintech, China). In brief, formalin-fixed, paraffin-embedded tissues were cut into 5-μm thickness and subjected to deparaffinization and rehydration. Following antigen retrieval, tissue sections were incubated with primary antibody overnight at 4 °C. After washed with PBS, sections were incubated with biotinylated secondary antibody for 1 h at room temperature. A DAB kit was employed as the chromogen and slides were counterstained with hematoxylin.
After deparaffinization, the sections were heated in EDTA for antigen retrieval and then blocked with BSA for 30 min at room temperature. The sections were then incubated with two kinds of primary antibodies at 4 °C overnight in a humidified chamber: CD16 antibody (1:5000, Servicebio, China) and S100A8/A9 antibody (1:5000, Abcam, UK). The next day, the sections were incubated with secondary antibodies for 50 min at room temperature. The secondary antibodies used were goat anti-rabbit Cy3 and goat anti-rabbit iF647 (both from Servicebio, China, diluted at 1:300). The nuclei were stained with DAPI for 10 min.
After deparaffinization, the sections were heated in EDTA for antigen retrieval and then blocked with BSA for 30 min at room temperature. The sections were then incubated with two kinds of primary antibodies at 4 °C overnight in a humidified chamber: CD16 antibody (1:5000, Servicebio, China) and S100A8/A9 antibody (1:5000, Abcam, UK). The next day, the sections were incubated with secondary antibodies for 50 min at room temperature. The secondary antibodies used were goat anti-rabbit Cy3 and goat anti-rabbit iF647 (both from Servicebio, China, diluted at 1:300). The nuclei were stained with DAPI for 10 min.
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