- Wash the slides 8 min in PBS and incubate them for 8 min in
PBST. Transfer the slides into an immunostaining system, and
add PBST to prevent drying. - Add blocking solution and incubate for 30 min at room tem-
perature (seeNote 59). - Wash 25 min with PBST.
- Add the primary anti-CD31 antibody diluted in blocking solu-
tion, and incubate for 2 h at room temperature or longer times
at 4C. - Wash 35 min with PBST.
- Add the fluorescent-labelled secondary antibody diluted in
blocking solution, and incubate for 60 min at room tempera-
ture in the dark. - Wash 35 min with PBST and 25 min with PBS.
- Embed the slides with fluoromount-G and mount a glass cov-
erslip over the Matrigel section (seeNote 60). - Let the slides dry overnight at room temperature and store
them in slide storage boxes in the dark at 4C(seeNote 61). - Scan the sections at 50magnification at a laser scanning
microscope or 200 magnification with an epifluorescent
microscope, and allow stitching of single images (seeNote 62). - For analyzing open the stitched pictures in ImageJ or a compa-
rable analyzing software. - Manually define the plug area and set an automatic threshold
(“Otsu”) (seeFig. 4). - Analyze CD31-positive pixels in the total plug area.
Fig. 4Analysis of Matrigel plug sections with ImageJ. (a) The plug section is scanned at a 200magnification
with an epifluorescent microscope, stitched automatically (see the adjusted brighter insert), and loaded into
ImageJ. The plug area is defined manually, an automatic threshold is set using the “Otsu” method, and the
CD31-positive area is normalized to the whole plug area. (b,c) An enlarged section of the whole plug is shown
to illustrate the vessels before (b) and after (c) thresholding. Scale bars 1000μm(a) or 100μm(b,c)
Angiogenesis 531