Ba1003

Breast tissues from the rats were fixed in 4% paraformaldehyde at room temperature for 48–72 h. The breast tissue was embedded in paraffin, and 5 μm-thick paraffin sections were prepared. The pathological changes of breast tissues were observed under the light microscope following hematoxylin and eosin (HE) staining. For IHC staining, the paraffin sections were deparaffinized with xylene and 3% hydrogen peroxide for antigen retrieval at room temperature for 10 min. Then, the sections were incubated with primary antibodies against PTEN (1 : 100; ab31392; Abcam, CA, USA), PI3K p85 (1 : 200; ab189403; Abcam), and p-Akt (1 : 50; ab38449; Abcam) at 4°C overnight followed by incubation with the appropriate amount of biotin-conjugated goat anti-rabbit IgG (BA1003) or biotin-conjugated goat anti-mouse IgG (BA1001; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 30 min at 37°C. Next, the reaction was visualized using AEC (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and counterstained with hematoxylin. Positive immune staining was presented as brown or yellow granules in the cytoplasm or the nucleus.

Tumor samples were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin, and 5 μm thick paraffin sections were prepared. Sections were deparaffinized, rehydrated, and subjected to antigen retrieval by 3% hydrogen peroxide. Then, the sections were incubated with the primary antibodies against TIMP3 (ab39184; Abcam, Cambridge, USA) overnight at 4˚C. Primary antibodies were detected using a biotin-conjugated goat anti-rabbit IgG (BA1003; Wuhan Boster Biological Technology, Ltd.) at 37˚C for 30 min. Following rinsing with PBS three times, the sections were exposed to 3,3′-diaminobenzidine (DAB; OriGene Technologies, Inc.) for 5 min and counterstained with hematoxylin. Brown or yellow granules in the cytoplasm or nucleus were considered positive immune staining. A Nikon imaging system was used for image collection.