Ki-67 immunofluorescence staining was performed on 4 µm tissue slices of hair follicles as mentioned previously2 (link). After de-paraffinized and rehydrated process, slices were fixed in 4% paraformaldehyde supplemented with 0.1% Triton X-100 for 10 minutes, equilibrated in phosphate buffer saline for 15 minutes and blocked in 4% normal donkey serum for 60 minutes. The slices were then incubated with anti-ki67 mouse monoclonal antibody (Abcam, Cambridge, MA, USA) overnight at 4℃, followed by incubation with Alexa Flour 488 conjugated donkey anti-mouse IgG (Abcam) for 60 minutes. And then, fluorescent images were analyzed using a confocal microscope (Leica Microsystems GmbH, Bannockburn, IL, USA).
Alexa flour 488 conjugated donkey anti mouse igg
Manufactured by Abcam
Sourced in United States
Alexa Fluor 488 conjugated donkey anti-mouse IgG is a secondary antibody used for detection and visualization of mouse primary antibodies in various immunoassays and imaging techniques. The antibody is conjugated to Alexa Fluor 488, a fluorescent dye that allows for bright and photostable detection.
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2 protocols using alexa flour 488 conjugated donkey anti mouse igg
1
Ki-67 Immunofluorescence Staining of Hair Follicles
2
Immunohistochemical Analysis of Kidney Proteins
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Frozen sections were fixed in 4% paraformaldehyde in PBS and were made permeable using 0.5% Triton-X100 in PBS. All subsequent washes were performed with 0.1% Tween 20 in TBS. Sections were blocked in 5% horse serum for 60 min, followed by incubation overnight at 4°C with diluted primary antibody anti-Rhbg (1:200; Abcam, Cambridge, UK), anti-Rhcg (1:200; Abcam), anti-dystrophin (1:100; Abcam), and anti-CD31 (1:300; BioLegend, San Diego, CA). Secondary antibodies were Alexa Fluor 546-conjugated goat anti-rabbit IgG (1:300; Life Technologies), Alexa Flour 488-conjugated donkey anti-mouse IgG (1:300; Abcam), and Cy3-conjugated donkey anti-goat (1:300; Jackson ImmunoResearch, West Grove, PA) with DAPI (Life Technologies). Subsequently, the sections were incubated for 60 min at room temperature. After several washes, the sections were enclosed on a mounting medium (KPL, Gaithersburg, MD). The images were obtained under a microscope (BX-51, Olympus, Tokyo, Japan) and processed using image analysis software (Openlab, PerkinElmer, Waltham, MA).
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