Pcna primary antibody

Djulis was obtained from Sinfong Agritech Co. (Taipei, Taiwan). Iron (III) chloride hexahydrate, methylene blue, and acetic acid were purchased from Nacalai Tesque (Tokyo, Japan), Showa Chemicals (Tokyo, Japan), and Shimakyu Pure Chemicals (Osaka, Japan), respectively. The Bax primary antibody and Bcl-2 primary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase and the caspase-9 primary antibody were purchased from GeneTex (Irvine, CA, USA). The proliferating cell nuclear antigen (PCNA) primary antibody, goat anti-rabbit immunoglobulin G (IgG) secondary antibody, and peroxidase AffiniPure goat anti-mouse IgG were purchased from Abcam (Cambridge, UK), Southern Biotechnology (Birmingham, AL, USA), and Jackson ImmunoResearch (West Grove, PA, USA), respectively. The β-actin primary antibody, 1,2-dimethylhydrazine (DMH), N,N’-dimethyl-p-phenylenediamine, N,N’-dimethyl-m-phenylenediamine, Alcian blue, and the remaining chemicals were purchased from Sigma Chemical (St. Louis, MO, USA).

After 21 days of HeLa/NIH 3T3 or HeLa/hMBSC coinjection, mice were euthanized with pentobarbital, and tumor tissues were isolated and fixed in 4% paraformaldehyde, embedded in paraffin. Tissue was cut into 5 μm-thick sections. After deparaffinization and rehydration, the sections were rinsed in PBS and then incubated in a 3% H₂O₂ solution to block the endogenous peroxidase. After incubation with 5% BSA for 30 min to block nonspecific antibody-binding sites, the samples were stained with PCNA primary antibody (1 : 1000, mouse monoclonal, Abcam) at 4°C overnight. The samples were rinsed with PBS twice and incubated with a HRP-conjugated goat anti-mouse secondary antibody (MaiXin biotechnologies, China) followed by visualization with 3,3-diaminobenzidine tetrahydrochloride (MaiXin biotechnologies). Finally, the sections were stained with hematoxylin and examined under a light microscope.
Apoptosis was analyzed on paraffinic tumor tissue sections of HeLa/NIH 3T3- and HeLa/hMBSC-coinjected groups by TUNEL assay kit (Millipore, USA). Three sections were selected for each mouse and stained using the TUNEL assay kit following the manufacturer's protocol. Under the microscope, cells with dark-brown nuclei were marked as positive and counted in 10 randomly selected fields per nude mouse with a total of 4 nude mice in each group.