Multi-color immunofluorescence labelling method and imaging method
A technology of immunofluorescence and labeling method, applied in the field of immunofluorescence, which can solve the problems of color shifting and limited staining types, etc., and achieve the effect of clear colors
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Embodiment 1
[0046] In this embodiment, the frozen section of C57 / B6 mouse heart is taken as a sample, and the multicolor immunofluorescent labeling method of this embodiment is described, as follows:
[0047] 1. Pretreatment
[0048] 1. Frozen sections of 1C57 / B6 mouse hearts were fixed with 4% paraformaldehyde for 15 minutes, and then washed with PBS buffer three times, 5 minutes each time.
[0049] 1.2 After washing, the frozen sections were incubated at 37°C for 1.5h with blocking membrane permeabilization solution.
[0050] Wherein, the sealing permeation solution is goat serum, which contains 0.3% Triton-100.
[0051] 2. The first fluorescent labeling
[0052] 2.1 Primary antibody incubation: use mouse-derived Troponin T antibody (purchased from Abcam, specification 200 μg, catalog number ab8295) (diluted at a ratio of 1:10) and rabbit-derived α-SMA protein-binding α -SMA antibody (purchased from Abcam, specification 10ul, product number ab32575) (diluted at a ratio of 1:20) and fro...
Embodiment 2
[0124] This embodiment provides a multi-color immunofluorescence imaging method, which uses the sample prepared in Embodiment 1 as the observation object, and uses a confocal microscope (Leica SP5 confocal microscopy) for imaging. The specific operation method is as follows:
[0125] Confocal procedure:
[0126] Use 405 laser to illuminate the light of Dylight405 (take pictures in the range of 415-445nm);
[0127] Use a 405 laser to perform spectral scanning (scanning the wavelength range of 415-481nm, with 3nm as a scanning unit, and scan once every 1nm increase (the way of spectral scanning is the same)) (split Dylight405 and DAPI) (abandon Dylight405, Save DAPI's graph);
[0128] Use 405 laser to illuminate the light of Qdot625 (take pictures in the range of 600-660nm);
[0129] Use 488 laser to perform spectral scanning (scanning 500-548nm) (split AF488, PE-Cy7 and BODIPY FL);
[0130] Use 514 laser to perform spectral scanning (scanning 524-599nm) (split AF514, AF532, ...
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