Figure 4.

Optimization of collagen IV staining. We tested different antigen retrieval methods to improve the staining of blood vessels with anti-collagen IV. (a) Low pH heat-mediated antigen retrieval of collagen IV staining led to only light reactivity. (c) High pH heat-mediated antigen retrieval did not improve blood vessel staining. (b, d) Though the microvessel algorithm did detect some vessels, there were many lightly-stained vessels that were not identified (red arrows). (e) Pepsin-mediated retrieval for 10 minutes improved blood vessel staining, while maintaining a very low background. (f) The microvessel algorithm detected more vessels using this method, but still missed some that were lightly stained (red arrows). (g) Pepsin-mediated retrieval for 20 minutes further improved staining of blood vessels, both small and large, while retaining the low background. (h) The microvessel algorithm was able to detect all stained vessels in the region of interest, while minimizing detection of non-vessels. Each of the antigen retrieval methods tested yielded an increase in the number of vessels detected by the algorithm (i), as well as the vessel density (j). Red boxes indicate the region of interest analyzed by the microvessel algorithm, shown in the second panel for each antibody.