Anti ki67

The paraffin blocks of the xenografts were cut into 5-μm slices. For H&E staining, the slides were dewaxed and dyed with hematoxylin for 10 min, placed in hydrochloric acid alcohol differentiation solution, and subsequently stained with eosin for 1 min. For the IHC assay, endogenous peroxidase activity was blocked using 0.35% hydrogen peroxide in PBS. Subsequently, antigens were retrieved by microwaving, and non-specific binding was blocked with 5% bovine serum albumin. The prepared tissue slices were incubated with anti-SHP2 and anti-p-ERK (1:200; Cell Signaling Technology), anti-p-CREB (1:800; Cell Signaling Technology), and anti-Ki-67 (1:200; ZSBIO, Beijing, China) antibodies overnight at 4°C. Signals were detected using a DAB Detection kit (ZSBIO) and images were captured under an Axio Observer.A1 microscope (Zeiss). For Ki-67 and p-CREB nuclear staining, the “count small-cells” function of the Image-Pro Plus version 6.0 software (Media Cybernetics, Rockville, MD, USA) was used to count the positive cells. For p-ERK staining, the integrated optical density was measured using the “measure stain” function. At least six random fields in each section were measured and three sections were selected from each xenograft.

5 × 10⁵ cells were seeded on coverslips (Thermo Fisher Scientific), washed with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.4% Triton X-100 in PBS for 30 min at RT. After washing with PBS three times, the cells were blocked with 10% donkey serum (Jackson ImmunoResearch) in PBS for 1 hr at RT. The cells were then incubated with primary antibodies in 10% donkey serum at 4°C overnight. Afterwards, cells were washed with PBS and stained with secondary antibodies and Hoechst 33342 (Thermo Scientific) for 1 hr at RT. A Leica SP5 confocal system was used for imaging. Antibodies used in this study were as follows: anti-Ki67 (ZSGB-BIO, ZM0166), anti-53BP1 (Bethyl Laboratories, A300-273A), anti-FOXA2 (Cell Signaling Technology, 8186S), anti-SMA (Sigma, A5228), anti-TuJ1 (Sigma, T2220), anti-H3K9me3 (Abcam, Ab8898), anti-NANOG (Abcam, Ab21624), anti-OCT3/4 (Santa Cruz, sc-5279), anti-SOX2 (Santa Cruz, sc-17320), and anti-LAP2 (BD Bioscience, 611000), anti-HP1α (Cell Signaling Technology, #2616S), and anti-Lamin A/C (Santa Cruz, sc-376248).

Xenograft tumors were collected, and paraffin-embedded sections were prepared. For immunohistochemistry staining, tumor sections were deparaffinized in xylene and rehydrated in graded alcohols. Then, sections were treated with 3% hydrogen peroxide for 15 mins and incubated with 10% goat serum for 30 mins to decrease nonspecific binding. After washing three times in PBS, the sections were incubated with anti-Ki67 (1:500, ZSGB-BIO, China, ZM-0166) at 4°C overnight, followed by incubation with peroxidase labeled secondary antibody at room temperature for 30 mins. The nuclei were counterstained with hematoxylin for 5 min, dehydrated, and mounted for microscopic examination.

For immunofluorescence analysis, cells seeded on coverslips (Thermo Fisher Scientific) with a suitable confluence were fixed in 4% PFA for 15 min, washed with PBS at least three times, permeabilized using 0.4% Triton X-100 in PBS for 30 min, and then blocked with 10% donkey serum (Jackson ImmunoResearch) in PBS for 1 h at room temperature. Cells were then incubated with indicated primary antibodies in 10% donkey serum in PBS at 4°C overnight. Cells were washed at least three times with PBS and incubated with secondary antibodies and Hoechst 33342 (Invitrogen, H3570) at room temperature for 1 h, then washed at least three times with PBS. Finally, the coverslips were mounted using mounting medium (Vector Labs), and images were captured with a Leica SP5 confocal system.
Antibodies used for immunofluorescence analysis are as follows: anti-SOX2 (Santa Cruz, sc-17320), anti-OCT4 (Santa Cruz, sc-5279), anti-NANOG (Abcam, ab21624), anti-Ki67 (ZSGB-BIO, ZM-0166), anti-γH2AX (Millipore, 05-636), anti-53BP1 (BETYHL, A300-273A), anti-Lamin B1 (Abcam, ab16048), anti-FLAG (Sigma, F1804), anti-H3K9me3 (Abcam, ab8898), anti-Lamin A/C (Santa Cruz, sc-376248), anti-KAP1 (Abcam, ab10483), anti-HP1α (Cell Signaling Technology, 2616), Alexa 488 donkey anti-mouse IgG (Invitrogen, A21202), Alexa 568 donkey anti-rabbit IgG (Invitrogen, A10042) and Alexa 647 donkey anti-goat IgG (Invitrogen, A21447).