Immunohistochemical Analysis of Rat Lung Tissue

Paraffin-embedded rat lung tissue was sectioned, dewaxed, and rehydrated as described previously [22 (link)]. Hematoxylin and eosin (H&E) staining was performed according to the operation manual of a hematoxylin and eosin staining kit (Beyotime, CHN). IHC was performed as described [23 (link)]. After blocking nonspecific protein binding, the sections were treated with mouse anti-α-SMA (1:500, Boster, Wuhan, CHN), anti-p-PI3K/anti-PI3K (1:100, Abcam, USA), anti-p-Akt/anti-Akt (1:200, Abcam, UK), anti-p-mTOR/anti-beta-actin (1:100, Abcam, UK), anti-p-PKC/anti-PKC (1:250, Cell Signaling, USA), or anti-p-p44/42/anti-p44/42 antibodies (1:250, Cell Signaling, USA) followed by incubation for 2 h with goat anti-rabbit secondary antibodies (HPR, Boster, CHN); then, ABC horseradish peroxidase reagent (Boster, CHN) was added for 30 min at room temperature. The secondary antibody controls (negative control) were incubated in PBS instead of the primary antibody. Specimens were observed under a fluorescent inverted microscope (IX73-A22FL/PH; Olympus Corporation, Japan), and images were captured by using the Image-Pro Plus software (version 6.0). The average optical density (IOD/area) was used as a semiquantitative index for analysis of the positive signals.