Alexa fluor 488 conjugated goat anti rabbit igg secondary antibody

Manufactured by Abcam

Sourced in United Kingdom

Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody is a fluorescently labeled secondary antibody used to detect and visualize rabbit primary antibodies in various immunoassays and imaging techniques.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Anti ldlr antibody, by Abcam (1 mentions) Dil ldl, by Biomedical Technologies (1 mentions) Facscan, by BD (1 mentions) Tris edta buffer solution, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Rabbit anti mouse cd31 antibody, by Abcam (1 mentions) Fluorescence microscope, by Olympus (1 mentions) Lsm 800, by Zeiss (1 mentions) Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions) Axio scan z1, by Zeiss (1 mentions) Colibri 7, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole dihydrochloride dapi, by Abcam (1 mentions)

7 protocols using alexa fluor 488 conjugated goat anti rabbit igg secondary antibody

Quantifying LDLR and Cholesterol Uptake

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Cell surface LDLR was detected by flow cytometry. Following treatment, LM3 cells were detached from the plates by scraping and washed with phosphate-buffered saline (PBS) containing 5% bovine serum albumin for 30 minutes at room temperature. Cells were then washed with PBS, incubated with an anti-LDLR antibody (Abcam) at room temperature for 1 hour, and then incubated with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Abcam) for 30 minutes at room temperature. Background fluorescence (control) was quantified using cells incubated with an isotype rabbit IgG antibody (Abcam) for 1 hour and Alexa Fluor 488-conjugated goat anti-rabbit IgG for 30 minutes. The cells were washed and resuspended in PBS, and then analyzed by flow cytometry using an FL1 emission filter (FACScan, BD Biosciences, San Jose, CA, USA). The data were analyzed using the FlowJo software.
Dil-LDL (Biomedical Technologies Inc., USA) uptake was representative of total cellular cholesterol uptake. Following treatment of LM3 cells, the media was exchanged to serum-free and incubated with 10 ug/mL Dil-LDL at 37°C for 12 hours. The cells were detached with trypsin, washed, and the resuspended in PBS for flow cytometry analysis using an FL2 emission filter (FACScan, BD Biosciences, San Jose, CA, USA). Data were analyzed using the FlowJo software (CA, USA).

He M., Hou J., Wang L., Zheng M., Fang T., Wang X, & Xia J. (2017). Actinidia chinensis Planch root extract inhibits cholesterol metabolism in hepatocellular carcinoma through upregulation of PCSK9. Oncotarget, 8(26), 42136-42148.

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Immunohistochemical Analysis of NF-κB Pathway

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Immunohistochemical staining was performed using the avidin-biotin-peroxidase technique. Paraffin-embedded tumor tissue sections (5 µm) were fixed with 10% paraformaldehyde for 2 h at 37°C were prepared and subsequently deparaffinized in xylene and washed in PBS with Tween-20 (1%) three times at room temperature, followed by epitope retrieval using Tris-EDTA buffer solution (pH 9.0; Sigma-Aldrich; Merck KGaA; cat. no. SRE0063) for 60 min at 65°C. The paraffin sections were subjected to hydrogen peroxide (3%) for 10–15 min, and were subsequently blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 10–15 min at 37°C. Finally, the sections were incubated with rabbit anti-mouse p65 (1:1,000; cat. no. ab16502), Ikk-β (1:1,000; cat. no. ab17870), IκBα (1:1,000; cat. no. ab16502; all Abcam) t 4°C for 12 h. All sections were washed three times and incubated with Alexa Fluor^® 488-conjugated goat anti-rabbit IgG secondary antibody (1:1,000; cat. no. ab150077; Abcam) for 1 h at 37°C and were observed in six random fields of view under a fluorescence microscope at ×40 magnification.

Zhao X, & Wang X. (2018). Candesartan targeting of angiotensin II type 1 receptor demonstrates benefits for hypertension in pregnancy via the NF-κB signaling pathway. Molecular Medicine Reports, 18(1), 705-714.

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Quantifying Microvessel Density in Tumor Sections

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Tumor paraffin sections (8 μm) were incubated with Tris/EDTA buffer for 30 min after hydrated. The sections were treated and blocked with PBS containing 0.3% Triton X- and then primarily incubated with rabbit anti-mouse CD31 antibody (1:100, Abcam, Cambridge, UK) at 4°C overnight. After washing, the sections were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:50, Abcam) for 2 h. For quantitative measurements of MVD, five slides, with each slide containing four fields (×200), were captured under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) and analyzed using ImageJ software (NIH, Bethesda, MD, US).

Liang C., Yuan X., Shen Z., Wang Y, & Ding L. (2022). Vorolanib, a novel tyrosine receptor kinase receptor inhibitor with potent preclinical anti-angiogenic and anti-tumor activity. Molecular Therapy Oncolytics, 24, 577-584.

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Immunofluorescence Analysis of Tight Junction Proteins and Inflammatory Markers in Caco-2 Cells

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Caco-2 cells were cultured in 12-well plates to assay the relative intensity of ZO-1, occluding, TLR4, and NF-κB p65 through immunofluorescence. After the corresponding treatments, the cells were washed with sterile PBS three times for 5 min and fixed with 4% stationary liquid at 4 °C for 10 min, then washed with sterile PBS three times for 5 min. Then, 600 µL/well of PBST (0.5% Triton X-100) was added for 10 min, washed once with sterile PBS for 5 min, and blocked with 10% goat serum for 1 h at room temperature. Cells were incubated with ZO-1(1:500), Occludin (1:200), TLR4 (1:100), and NF- kB p65 (1:100) overnight at 4 °C. The cells were washed with PBST three times for 5 min. Then, 400 μL/well of Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Cat# ab150077, abcam, 1:500) was added for 1 h at 37 °C in the dark and washed with PBST three times for 5 min. Finally, 400 μL/well of Hoechst 33258 was added to counterstain cell nuclei for 5 min. The cells were imaged with a laser scanning confocal microscope (ZEISS, LSM800, Oberkochen, Germany), and the relative fluorescence intensity was acquired using Image J (V1.8.0, National institutes of health, Bethesda, USA).

Zheng J., Ahmad A.A., Yang Y., Liang Z., Shen W., Feng M., Shen J., Lan X, & Ding X. (2022). Lactobacillus rhamnosus CY12 Enhances Intestinal Barrier Function by Regulating Tight Junction Protein Expression, Oxidative Stress, and Inflammation Response in Lipopolysaccharide-Induced Caco-2 Cells. International Journal of Molecular Sciences, 23(19), 11162.

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Immunofluorescence Staining of TGFβRII in Lung Sections

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Lung sections were sectioned at 5 μm thickness, deparaffinized, and rehydrated in a graded ethanol series. Antigen retrieval was performed using a citrate buffer solution, followed by incubation with 2% normal goat serum (Jackson ImmunoResearch Laboratory, Inc., West Grove, PA, USA) and rabbit anti-TGFβRII primary antibody (catalog no: ab18683; Abcam, 1:200, UK). Sections were then incubated in the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (catalog no: ab150085; Abcam, UK) and DAPI (catalog no: D9542; Sigma-Aldrich, USA) for immunofluorescence detection. Images were captured using a slidescanner (ZEISS^® Axio Scan. Z1), connected to an LED light source (ZEISS^® Colibri 7), with an imaging software (ZEN Blue 2.3).

Seenak P., Kumphune S., Prasitsak T., Nernpermpisooth N, & Malakul W. (2022). Atorvastatin and ezetimibe protect against hypercholesterolemia-induced lung oxidative stress, inflammation, and fibrosis in rats. Frontiers in Medicine, 9, 1039707.

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Immunofluorescence Verification of Neutralization

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Immunofluorescence was used to further verify the neutralization test. Vero cells in plate were washed with phosphate-buffered saline (PBS), fixed in 80% precooled acetone (Sigma-Aldrich, USA) for 30 min, then washed and blocked in 1% bovine serum albumin at room temperature (19-21°C) for 30 min, and then incubated with anti-Spike RBD Rabbit monoclonal antibodies (mAbs) (1:1000; Sino Biological) at 4°C overnight. The plates were washed and added with Alexa Fluor488^®-conjugated Goat Anti-rabbit IgG secondary antibody (1:1500; Abcam, Cambridge, UK) at room temperature for 2 hours. 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (2 µM; Abcam) was used to stain the nuclei (Figure S1).

Shi D., Weng T., Wu J., Dai C., Luo R., Chen K., Zhu M., Lu X., Cheng L., Chen Q., Liu F., Wu Z., Wu H., Jin C., Guo M., Chen Z., Wu N., Yao H, & Zheng M. (2021). Dynamic Characteristic Analysis of Antibodies in Patients With COVID-19: A 13-Month Study. Frontiers in Immunology, 12, 708184.

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Immunofluorescence Assay for Ki67 in dPASMCs

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dPASMCs were seeded on coverslips in 24-well plates for immunofluorescence assay. After starved for 24 h prior to indicated treatments, dPASMCs were fixed in 4% paraformaldehyde for 20 min and rinsed with PBS three times. Subsequently, dPASMCs were permeabilized with 0.3% Triton X-100 at room temperature for 10 min, and then blocked with 5% non-fat milk for 30 min. The primary antibody against ki67 (1:250, Abcam, Cambridge, UK) was incubated overnight at 4 °C. After washing three times with PBS, dPASMCs were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:500, Abcam) for 30 min at 37 °C in the dark, then, washed three times with PBS before being incubated with 4′,6-diamidino-2-phenylindole (DAPI) (1 µg/mL) (Sigma-Aldrich, St. Louis, Missouri, USA) for 5 min. Finally, images were captured under a fluorescence microscope (Leica DMi8, Wetzlar, Germany) and quantitative analysis was performed using Image J analysis software.

He T., Zhang J., Qiao T., Zhang Z., Han H., Yang C., Chen Y., Ruan Y, & Meng L. (2022). Compensatory roles of Protein Related to DAN and Cerberus (PRDC) decrease in pulmonary arterial hypertension. International Journal of Biological Sciences, 18(6), 2372-2391.

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