Ix73 a22fl ph

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.

For the immunofluorescence assay, the lung sections were treated with rabbit anti-RELM-β polyclonal antibodies (1:200, Abcam, UK) and mouse anti-α-SMA antibody (1:500) or the corresponding vehicle overnight at 4 °C. Then, the sections were incubated with FITC-conjugated AffiniPure goat anti-mouse IgG (H + L) (1:50, Boster, CHN) (excitation: 495 nm, emission: 525 nm) and Cy3-conjugated AffiniPure goat anti-mouse IgG (H + L) (1:50, Boster, CHN) (excitation: 554 nm, emission: 568 nm) for 45 min at room temperature in the dark. Finally, the sections were washed in PBS and mounted with Vectashield hardset mounting medium with 4′,6-diamidino-2-phenylindole dilactate (DAPI, Vector Laboratories, USA). Specimens were observed under a fluorescent inverted microscope (IX73-A22FL/PH; Olympus Corporation; light source: UHP) attached to a CCD digital camera (512B Cascade, Roper Scientific, Tucson, AZ). Blue (WU) and green filters (WIBA) were used to detect FITC (green) and Cy3 (red) signals, respectively. The objectives used were 20 × and 40 × . Images were captured by using the Image-Pro Plus software (version 6.0) and colorized with Adobe Photoshop CS6 software (Adobe Systems, Inc., San Jose, CA).

At 21 days, all mice were put to death by cervical dislocation, and the tumors and lung organs were taken out from BALB/c nude mice. Hypoxia-induced PAH and normal lung tissue were taken out from the SD rats and mice. All the organs’ sections were treated with G6PD antibodies (1:1000, Abcam, USA) overnight at 4° C. Then, the sections were exposed to a FITC-conjugated secondary antibody (1:1000) for 2 h. Finally, samples were visualized using a fluorescent inverted microscope (IX73-A22FL/PH; Olympus Corporation, Japan; light source: UHP).