Ab6566

Manufactured by Abcam

Sourced in United States, China

Ab6566 is a laboratory reagent manufactured by Abcam. It is used as a component in scientific research and experiments. The core function of this product is to facilitate specific chemical or biological analyses, but no further details about its intended use are provided.

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Lab products found in correlation

5 protocols using ab6566

Immunohistochemical Analysis of Kidney Markers

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Sections were rehydrated and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Samples were blocked with 3% BSA in PBS and incubated with primary antibodies (anti-Six2, 1:500; 11562-1-AP Proteintech, Chicago, IL, anti-Collagen IV, 1:500; ab6566 Abcam, anti-Pax2; 1:500; ab79389, Abcam). Samples were then incubated with secondary antibodies, Alexa Fluor 594 conjugate and Zenon IgG labeling Kit Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA), for 1 h. Nuclear counterstaining was performed using DAPI or DRAQ5 (BioStatus, Leicestershire, UK), followed by mounting in Prolong-Gold (Thermo Fisher Scientific). Images were obtained by confocal (FV1000; Olympus) or standard (IX-71, DP-73; Olympus) microscopy.

Kirita Y., Kami D., Ishida R., Adachi T., Tamagaki K., Matoba S., Kusaba T, & Gojo S. (2016). Preserved Nephrogenesis Following Partial Nephrectomy in Early Neonates. Scientific Reports, 6, 26792.

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RPE Cell Culture and Analysis

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The RPE cells isolated from the eyes of CXCR5 KO mice and WT-control mice were seeded and cultured on a cellulose matrix with a pore size of 0.45 μm (R9NA09917, Millicell, Merk Millipore) for 4 weeks in complete N1 media. Before the fixation, the RPE cell cytoplasm was tagged with calcein AM (Thermo Fisher; C1430; 1 μg/ml) in culture for 30 min. Cels were fixed in 2% paraformaldehyde (VVR Life Science) for 10 min and blocked with 2.5% normal goat serum for 1 h at RT. Cells were fixed with, blocked with BSA as described above, and incubated overnight at 4 °C with β-amyloid and APOE antibodies (Table 1), followed by detection with Cy5 conjugated anti-rabbit (A10523, 1:1000; Thermo Fisher) and anti-goat (ab6566; 1:1000; Abcam) secondary antibody. Following washing with PBS-T three times for 15 min, membranes were embedded in OCT compound snap-frozen using liquid nitrogen-cooled isopropanol, and 5 μm cross-sections were prepared. The resulting samples were mounted with ProLong Diamond antifade reagent and imaged.

Lennikov A., Mukwaya A., Saddala M.S, & Huang H. (2020). Deficiency of C-X-C chemokine receptor type 5 (CXCR5) gene causes dysfunction of retinal pigment epithelium cells. Laboratory investigation; a journal of technical methods and pathology, 101(2), 228-244.

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Protein Expression Analysis in NRK-52E Cells

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NRK-52E cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Suzhou, China) to extract the total protein. The protein was separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Sigma-Aldrich, St. Louis, USA). Then, the membranes were placed in 5% milk for 2 h, incubated with 1000 times diluted antibodies of Col IV (ab6566), α-smooth muscle actin (α-SMA, ab32575), p62 (ab109012), microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/I (ab128025), Beclin 1 (ab62557), phosphatase and tensin homolog deleted on chromosome ten (PTEN, ab32199), phosphatidylinositol 3-kinase (PI3K, ab189403), phosphorylated (p)-PI3K (ab182651), total (t)-protein kinase B (t-AKT, ab8805), p-AKT (ab38449), p-mTOR (p-mTOR, ab109268), and t-mTOR (ab63552, Abcam, Cambridge, USA) at 4°C overnight; then anti-mouse immunoglobulin G (IgG) antibody (1:2000; ab150113, Abcam, Cambridge, USA) was added and the membranes were incubated for 1 h. The bands were analyzed by an imaging system (Bio-Rad, Hercules, USA) and ImageJ software (NIH Image, Bethesda, USA).

Guo L., Tan K., Luo Q, & Bai X. (2020). Dihydromyricetin promotes autophagy and attenuates renal interstitial fibrosis by regulating miR-155-5p/PTEN signaling in diabetic nephropathy. Bosnian Journal of Basic Medical Sciences, 20(3), 372-380.

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Immunofluorescent Staining of Gut Hormones

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7 μm wax embedded sections were stained for PYY using primary polyclonal rabbit anti-PYY (1:1000, Abcam, ab22663), with secondary FITC-conjugated chicken anti-rabbit antibody (1:200, Abcam, ab6825). Staining for GLP-1 was carried out using primary polyclonal mouse anti-GLP-1 with secondary TRITC-conjugated goat anti-mouse (1:200, Abcam, ab5867). BrdU was stained for using primary monoclonal mouse anti-BrdU (1:100, Sigma, 13843420001) with secondary TRITC-conjugated polyclonal goat F(Ab) anti-mouse (1:200). 5HT was stained for using primary goat anti-5HT (1:500 Abcam, ab66047) with polyclonal donkey anti-goat (1:50, Abcam, ab6566). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) in Fluroshield (Abcam). Immunofluorescent images were acquired on a Carl Zeiss Axiovert 100S TV microscope. The operator performing the cell-counting was blinded to the groupings.

Brooks L., Viardot A., Tsakmaki A., Stolarczyk E., Howard J.K., Cani P.D., Everard A., Sleeth M.L., Psichas A., Anastasovskaj J., Bell J.D., Bell-Anderson K., Mackay C.R., Ghatei M.A., Bloom S.R., Frost G, & Bewick G.A. (2016). Fermentable carbohydrate stimulates FFAR2-dependent colonic PYY cell expansion to increase satiety. Molecular Metabolism, 6(1), 48-60.

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Multicolor Immunofluorescence Imaging of LSG

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The molecules of CD8, IFN-γ, and TNF-α were detected by immunofluorescence in the paraffin-embedded sections of LSG tissue. The same method was used as IHC described above, but fluorescence-conjugated secondary antibodies were used and DAPI was used as a chromatin counterstain. The primary antibodies were a mouse monoclonal antibody to CD8α (dilution 1:50, ab17147; Abcam), a rabbit monoclonal antibody to IFN-γ (0.02 mg/mL, MAB2853; Biotechne R&D Systems), and a goat polyclonal antibody to TNF-α (0.02 mg/mL, PA5-46945; Invitrogen). The secondary antibodies were goat anti-mouse IgG (FITC conjugated, dilution 1:100, Xi’an Zhuangzhi, China), goat anti-rabbit IgG (Cy3 conjugated, dilution 1:800, Xi’an Zhuangzhi, China), donkey anti-goat IgG with (Cy5 conjugated, dilution 1:2000, ab6566; Abcam). The stained slides were digitized with a Pannoramic scanner (3DHISTECH, Budapest, Hungary) and were analyzed by corresponding SlideViewer software.

Li H., Zhou Y., Wang P., Wang Y., Feng Y., Zhang Y, & Wu Z. (2023). Alterations of CD8+ T cells in the blood and salivary glands of patients with primary Sjögren’s syndrome. Clinical Rheumatology, 42(5), 1327-1338.

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