Ab32136

The rats were deeply anesthetized and perfused with 0.9% saline and 4% paraformaldehyde. The isolated brain was fixed in 4% paraformaldehyde for 24 h and dehydrated using sucrose (20%, 30%) for 24 h. Then, it was embedded in an OCT compound for cryosection and cut into 10 μm thick coronal sections. Next, fluorescent immunolabeling was performed according to the standard indirect technique as described previously. Primary antibodies used included PTEN (ab79156, 1:100, Abcam, Cambridge, MA, USA), β-APP (ab32136, 1:100, Abcam, Cambridge, MA, USA) and β III Tubulin (sc-51670, 1:200, Santa Cruz, Dallas, TX, USA). After washing three times in PBS, Dylight 488 conjugated Goat Anti-Rabbit IgG (A23220, 1:100, Abbkine, CA, USA) and Dylight 594 conjugated Goat Anti-Rat IgG (A23440, 1:100, Abbkine, CA, USA) were added to PBS containing 1% BSA for 1 h. In the final wash, 6-diamidino-2-phenylindole (DAPI) (28718-90-3, 1:100, Abbkine, CA, USA) was added and used as a counterstain for the nucleus. A ZEISS confocal laser scanning microscope was used to obtain fluorescence images (ZEISS LSM700, GER). PTEN⁺/β-APP⁺, in which positive cells were counted in 5 randomly selected regions (100×) of each slice using ImageJ software. Immunofluorescence staining was performed for five brains.

A spinal cord segment, 0.5 cm in length, from where the injured site was centered
was used for protein quantification using western blotting. Proteins of the
nucleus and cytosol were extracted using the NE-PER Nuclear and Cytoplasmic
Extractions Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the
manufacturer’s instructions. The protein mixture extracted from each segment was
separated via electrophoresis and transferred to the polyvinylidene difluoride
membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were first
incubated with PFKFB3 (1:5,000, ab181861, Abcam, Cambridge, UK), Bax (1:1,000,
ab32503, Abcam), Bcl-2 (1:500, ab59348, Abcam), cleaved caspase-3 (CC-3, 1:500,
ab13847, Abcam), p-CDK1 (1:1,000, ab201008, Abcam), p27 (1:5,000, ab32034,
Abcam), H3 (1:5,000, ab1791, Abcam), myelin basic protein (MBP) (1:1,000,
ab209328, Abcam), APP (1:1,000, ab32136, Abcam), β-actin (1:5,000, ab8226,
Abcam), and p-p27 (1:1,000, ab75908, Abcam) antibodies at 4°C overnight,
respectively, and incubated with the secondary antibodies (1:10,000, ZB-2301 or
ZB-2305, Zhongshan Gold Bridge, Beijing, China) at 25°C for 1 h. The proteins
were detected using an ECL kit (Immobilon, Millipore, Billerica, MA, USA). The
gray values of each band were calibrated according to the internal reference and
compared to those of the sham group to acquire a relative value.