Alexa fluor 488 donkey anti rabbit lgg

Cells or tissue sections were fixed in 4% paraformaldehyde. After washing 3 times with PBS for 3 min each time, the samples were incubated in 10% normal donkey serum in PBS for 20 min, and then incubated with MSI2 (Abcam, ab76148), CD44v6 (Abcam, ab78960) or Notch1 (Abcam, ab52627) primary antibodies in PBS at 4 °C for overnight. Incubated the samples with fluorochrome-conjugated secondary antibodies (1:400, Alexa Fluor®488 donkey anti-rabbit lgG, or Alexa Fluor®594 donkey anti-rabbit lgG, or Alexa Fluor®594 donkey anti-mouse lgG, life technologies) for 30 min, and subsequently incubated with DAPI. The images were observed and collected under fluorescence microscope. Three random fields in 200× were selected for quantification. ImageJ software was used to analyze the fluorescence intensity of CD44v6, MSI2 and Notch1. The experiments were repeated independently three times.

Briefly, after the sections were mounted and dewaxed, antigen retrieval was performed by a high-temperature, high-pressure method. After blocking in donkey serum (AntGene, ANT051), the sections were probed with primary antibodies, including anti-CD 41 antibody (diluted 1:500, Abcam, ab181582) and anti-CD 31 antibody (diluted 1:1000, Abcam, ab24590), overnight at 4 ℃. The sections were incubated with Alexa Fluor 488 donkey anti-rabbit lgG (diluted 1:400, Life Technologies, A21206) for 30 min at 37 ℃, and DAPI (1:500, Roche, 216276) was used to stain the nuclei. The platelets and thrombi were stained green and scanned by a fluorescence scanner (pannoramic Viewer MIDI II). Green foci with diameters less than 8 µm dismissed at suitable brightness and contrast in Pannoramic Viewer software. The maximum density (MD), defined as the maximum area of green foci in a fixed area of 750 µm × 750 µm at 100 × , and the thrombosis index (TI), defined as the ratio of the total green-stained area to the total stained area, were used to estimate the myocardial thrombus burden.