Rabbit mmp2

After cryosection, 2 μm thick brain sections were mounted onto glass slides. Sections were washed with PBS (pH 7.4), followed by antigen retrieval using sodium citrate buffer, pH 6, at 95°C for 25 min in a water bath. Nonspecific staining in sections was blocked using 10% donkey serum in 0.1% Tween 20-PBS for 1 h at room temperature. Primary antibodies were added overnight at a dilution of 1 : 350 for goat Iba-1 (Abcam), 1 : 1,000 for rabbit MMP-2 (Abcam) at 4°C. Alexa 488-conjugated donkey anti-goat IgG (1 : 200, Jackson Lab, Suffolk, United Kingdom) or Cy3-conjugated donkey anti-rabbit IgG (1 : 200, Jackson Lab) was subsequently applied. The nuclei were counterstained with DAPI (Sigma-Aldrich). Images were taken using a microscope (Axio Imager Z1, Carl Zeiss, China). This part was similar with the methods of Hu et al. [32 (link)].

Western Blotting was performed as described previously [11 (link), 30 (link)]. The total proteins of treated and tranfected cells were immunoblotted with rabbit p300, ERα-36 (Abcam), mouse phosphorylated STAT3 (Abcam), rabbit STAT3 (Abcam), rabbit MMP2(Abcam), mouse MMP9 (Abcam) and mouse p53, β-tubulin, and GAPDH (Santa Cruz) antibodies overnight at 4 °C, and then incubated with IR Dye™-800 conjugated anti-rabbit secondary antibodies and IR Dye™-680 conjugated anti-mouse secondary antibodies (Li-COR) for 30 min at room temperature (RT). The specific proteins were visualized by Odyssey™ Infrared Imaging System (Gene Company). GAPDH expression was used as an internal control to show equal loading of the protein samples.

Immunocytochemistry assays were performed as described previously [11 (link), 31 (link)]. The cells after treatment were fixed in 4% paraformaldehyde for 15 min, and then blocked with normal goat serum for 20 min. Then, rabbit MMP2(1:200 dilution, Abcam), and mouse MMP9(1:200 dilution, Abcam) antibodies were added and incubated in a humid chamber over night. After washing with PBS twice, cells were incubated with appropriate secondary antibodies (fluorescein isothiocyanate (FITC)-goat anti-rabbit or anti-mouse IgG, SantaCruz) for 30 min at 37 °C. After washing with PBS, the samples were observed under laser scanning confocal microscope (Olympus). 4′,6-diamidino-2-phenylindole (DAPI) stain (blue) highlights the total nuclei.