For immunofluorescence, HaCaT cells (2×105) were plated into a confocal small dish, washed twice with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.5% Triton-X-100/PBS for 5 min and blocked with 1% BSA for 1 h at room temperature. The cells were then washed again with PBS twice, and stained with the appropriate primary antibodies: Anti-E-cadherin (1:200; cat. no. 3195; Cell Signaling Technology, Inc.); and anti-vimentin (1:100; cat. no. AF1975; Beyotime Institute of Biotechnology) overnight at 4°C. Followed by incubation with the appropriate secondary antibodies: Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1:250; cat. no. ab150077; Abcam); and Alexa Fluor 555-Labeled Donkey anti-Rabbit IgG (1:250; cat. no. P0179; Beyotime Institute of Biotechnology) for 1 h at 37°C, respectively. Subsequent to washing twice with PBS, Hoechst staining was performed at room temperature for 3 min and the fluorescence was visualized with a confocal microscope (magnification, ×400).
Alexa fluor 555 labeled donkey anti rabbit igg
Manufactured by Abcam
Sourced in United Kingdom
Alexa Fluor 555-labeled donkey anti-rabbit IgG is a secondary antibody used for detection in immunoassays. It is conjugated to the Alexa Fluor 555 fluorescent dye, which can be excited at 555 nm and emits light at 565 nm.
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3 protocols using alexa fluor 555 labeled donkey anti rabbit igg
1
Immunofluorescence Imaging of E-cadherin and Vimentin
2
Identifying Endocrine Cells and Enteric Neurons
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To determine the types of CXCL14 immunoreactive endocrine cells and enteric neurons, double immunofluorescence staining was performed using rat anti-somatostatin serum (NP-105 SSTrat; Protos Biotech Corporation, New York, N.Y., USA) diluted to 1:500 in PBS-BSAT. Immunoreactivities were visualized by Alexa Fluor 555-labeled donkey anti-rabbit IgG (Abcam, Cambridge, UK) for CXCL14 and Alexa Fluor 488-labeled donkey anti-rat IgG (Abcam) for somatostatin.
3
Immunohistochemical Analysis of Breast Cancer
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An immunohistochemical analysis was performed on 10 cases of fully invasive ductal carcinoma of the breast, including 7 cases with skin invasion, using archival samples for pathologic diagnoses that had been fixed in formalin and routinely processed for embedding in paraffin. The use of samples was approved by written informed consent from patients under a protocol authorized by the Institutional Review Board of our university. All sections were cut at a thickness of 4 mm and placed on silane-coated glass slides. Antigen retrieval was performed by Target Retrieval Solution (pH 6.0; S1699; Dako Japan, Tokyo, Japan) using an autoclave (121 C for 1 minute). Sections were treated with 10% goat or donkey serum before an incubation with primary antibodies overnight. After being washed, sections were treated with the secondary antibodies of Alexa Fluor 488-labeled goat anti-rabbit IgG and Alexa Fluor 555-labeled goat anti-mouse IgG, followed by nuclear staining with DAPI. When the double staining of the integrin av and b1 subunits was performed, Alexa Fluor 488-labeled donkey anti-goat IgG and Alexa Fluor 555-labeled donkey anti-rabbit IgG (Abcam) were used. Observations were performed under an epifluorescence microscope.
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