For hematoxylin and eosin (H&E) staining, the mouse lung tissues were fixed with 4% paraformaldehyde for 24 h. The paraffin-embedded tissues were cut in 5 µm thick sections. H&E staining was employed to assess the alterations in morphology in these tissues using an optical microscope, and different fields were subsequently photographed.
For immunohistochemistry (IHC) analysis, paraffin-embedded tumor and lung tissues were deparaffinized and rehydrated as described [17 (link)]. After antigen retrieval and blocking, sections were incubated with anti-CD31 (1:50, ab182981, Abcam, Cambridge, UK), anti-TIMP2 (1:200, ab180630, Abcam) or anti-TIMP3 (1:100, ab213063, Abcam) antibody at 4 °C overnight, followed by the incubation with secondary antibody. The immunoreactivity was visualized by using DAB substrate (Thermo Fisher Scientific).
For immunohistochemistry (IHC) analysis, paraffin-embedded tumor and lung tissues were deparaffinized and rehydrated as described [17 (link)]. After antigen retrieval and blocking, sections were incubated with anti-CD31 (1:50, ab182981, Abcam, Cambridge, UK), anti-TIMP2 (1:200, ab180630, Abcam) or anti-TIMP3 (1:100, ab213063, Abcam) antibody at 4 °C overnight, followed by the incubation with secondary antibody. The immunoreactivity was visualized by using DAB substrate (Thermo Fisher Scientific).
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