Ab16901

The tissue microarray chip and slides were deparaffinized and rehydrated using xylene and a graded series of alcohols. Endogenous peroxidases were removed through incubation with 3% H₂O₂ for 20 min at 37 °C. Then, slides were autoclaved in 10 mM sodium citrate (pH 6.0) for 30 min to unmask antigens, washed in TBS (pH 7.4) three times, and incubated with primary antibodies against GPAA1 (1:300, Bioss, bs-13496R), ERBB2 (1:300, abcam, ab16901), p-AKT (1:100, Cell Signaling Technology (CST), #4060), Ki-67 (1:400, Bioss, bs-23105R), MMP2 (1:300, abcam, ab97779), MMP9 (1300, abcam, ab38898), the urokinase receptor (UPAR) (1500, abcam, ab218106) at 4 °C overnight. Slides were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and signal amplification and detection were conducted using DAB according to the manufacturer’s instructions (CST). Finally, images were acquired with a Nikon microscope.

Aldh1l1-eGFP were perfused with PBS followed by 4% PFA. Brains were postfixed in 4% PFA overnight at 4C, equilibrated in 30% sucrose, mounted in O.C.T. (Tissue-Tek) and sectioned into 10 µm thick sagittal cryosections. Sections were blocked with 10% goat serum in 0.1% PBST then stained with 1∶1000 chicken-anti-GFP (EMD Millipore AB16901), 1∶1500 rabbit-anti-Psmad1/5 (Cell Signaling 9516), 1∶300 rabbit-anti-ki67 (Cell Signaling 12075S) and 1∶2000 mouse-anti-GFAP (Sigma G3893) antibodies overnight at 4C. Alexa594, 568, 647 or 488 conjugated secondary antibodies were then added at 1∶1000 for 2 hrs at room temperature and sections were mounted in DAPI Fluoromount-G (Southern Biotech). Image acquisition was done at 20x on a Zeiss Axiocam microscope and expression quantified using ImageJ. Simultaneous staining with rabbit-anti-p-smad1/5 and rabbit-anti-ki67 was done using a sequential technique. P-smad1/5 staining and visualization with an Alexa568 conjugated secondary was completed first. Then, the sections washed repeatedly and stained with Alexa647 conjugated ki67. Co-localization of eGFP+ astrocytes with ki67 and p-smad1/5 at P3 was examined in 3 independent brains. P-smad1/5 colocalization with eGFP+ astrocytes was examined in two independent mouse brains for each postnatal time point. Significance was determined using a students t-test.