Zf 0311

The rats were sacrificed after hemodynamic measurements, and the lung and heart were quickly harvested and fixed in situ via the trachea cannula with buffered 4% formaldehyde, and embedded in paraffin. The sections were cut into 4-5 μm slices and were stained with streptavidin peroxidase and hematoxylin and eosin (H&E). For immunohistochemistry, 4-5 μm-thick cryosections or PAMSCs were first blocked with 5% goat serum (ab7481; Abcam, Cambridge, UK) for 30 min. The sections were then incubated with proliferating cell nuclear antigen (PCNA) (ab18197, Abcam) and CD31 (ab28364; Abcam). Subsequently, the 3, 3′- diaminobenzidine (DAB) dye was added to visualize the antibodies, and following washing of the tissue sections with phosphate-buffered saline (PBS) solution, the sections were observed and photographed under a microscope. For immunofluorescence, the sections or cells were incubated with rabbit polyclonal to smooth muscle α-actin (α-SMA, ab5694) or a nonspecific IgG antibody for 1 h at room temperature, which was followed by 1-h incubation in the dark with florescence isothiocyanate–conjugated goat anti-rabbit secondary antibody (ZF-0311; ZSGB-Bio Co., Beijing, China). Fluorescent images were taken with a Nikon Eclipse 90i microscope. Cells were fixed and permeabilized in a 50%.

Antibodies used and the sources are as follows: NFIB (ab186738, 1:1000 for WB), ORC1 (ab85830, 1:1000 for WB), CDC6 (ab188423, 1:1000 for WB), CDT1 (ab70829, 1:1000 for WB), MCM5 (ab76023, 1:1000 for WB), MCM6 (ab201683, 1:1000 for WB), MCM7 (ab52489, 1:1000 for WB), PCNA (ab29, 1:2000 for WB), H3 (ab1791, 1:2000 for WB) and BrdU (anti-CldU, ab6326) from Abcam; MCM2 (3619, 1:1000 for WB) from Cell Signaling Technology; ΟRC2 (sc-32734, 1:500 for WB), Geminin (sc-74456, 1:1000 for WB), POLD1 (sc-17776, 1:1000 for WB) and β-actin (sc-47778, 1:2000 for WB) from Santa Cruz Biotechnology; MCM4 (13043-1-AP, 1:1000 for WB), Cyclin E1 (11554-1-AP, 1:1000 for WB) and MCM3 (A1060, 1:1000 for WB) from Abclonal; NFIB (A303-566A, for CUT&Tag) and ORC1 (A301-892A, for CUT&Tag) from Bethyl Laboratories; c-myc-Tag (B1022, 1:2000 for WB), HA-Tag (B1021, 1:2000 for WB) and GAPDH (B1034, 1:2000 for WB) from Biodragon. FITC or TRITC-conjugated secondary antibodies (ZF-0311, ZF-0312, ZF-0316, ZF-0313, 1:100 for IF) from ZSGB-BIO.

After the imaging procedures, the solitary xenograft tumors were fixed in 4% buffered formalin. Hematoxylin and eosin (H&E) staining was performed on serial sections of 4 μm intervals for histological examination. The surface and inner samples from these samples were frozen and sectioned to detect TRAIL and EGFP expression, as previously described [28 (link)]. The sections were blocked with normal goat serum for 30 minutes (Zli-9021, ZSGB-BIO, Beijing) and then incubated simultaneously with a 1:100-diluted polyclonal rabbit anti-TRAIL primary antibody (AB2435, Abcam, U.S.A.) and a 1:20-diluted polyclonal mouse anti-EGFP primary antibody (AB16278, Abcam, U.S.A.). Our detection of TRAIL-MSCs was based on incubation with a FITC-conjugated goat anti-rabbit secondary antibody (ZF0311, ZSGB-BIO, Beijing) and a rhodamine-conjugated goat anti-mouse secondary antibody (ZF0313, ZSGB-BIO, Beijing).

Brain tissues were fixed in paraformaldehyde, embedded, and cut into sections (5 μm thickness). Then tissue sections were dewaxed in xylene and rehydrated in graded alcohols. After that, sections were incubated with 0.3% hydrogen peroxide to inactivate endogenous peroxidase activity. Antigen retrieval was achieved by incubating with 0.1 M sodium citrate (pH 6.0). After blocking with goat serum (Cat# ZLI-9022; Zhong Shan-Golden Bridge Biological Technology CO., Ltd., Beijing, China) for 30 min, sections were incubated with primary antibodies against TPH (1 : 300; ab46200; Abcam), ERβ (1 : 200; ab3577; Abcam), and SERT (1 : 1000; ab44520; Abcam) at 4°C overnight. After washing with PBS, secondary antibodies of FITC tagged goat anti-rabbit IgG (1 : 100; ZF-0311, ZSGB-bio, Beijing) were added and incubated for 30 minutes in the dark. After rinsing with PBS for 3 times, the sections were analyzed on a laser scanning confocal microscope (LSM510, Zeiss, Jena, Germany). Three different visual fields were randomly selected for each target brain area of each slice for photographing. The fluorescence intensity was quantified using Image-Pro Plus 6.0 (Media Cybenetics, Silver Spring, USA).

After fixed with 4% paraformaldehyde for 30 min, cells were permeabilized by 0.2% Trutib X-100 in PBS for 10 min. Blocked for 1 h with 3% BSA/PBS, cells were incubated with primary antibody (anti-myosin, 1:300, M4276, Sigma-Aldrich) overnight and then fluorescein-conjugated secondary antibody (ZF-0311, ZSGB-BIO, Beijing, China) at 1:100 for 1 h. Nuclei were stained with DAPI (62248, Thermo Scientific). Representative images from each slide were acquired by the inverted fluorescence microscope.