Rabbit anti mouse antibody

Total protein was extracted to be used in ELISA assays for mouse Nrf2 (MBS7612500, MyBioSource), IL-1β (ab197742; Abcam), iNOS (MAB9502, R&D Systems), tumor necrosis factor alpha (TNFα, ab208348; Abcam), IL-6 (ab222503, Abcam), arginase 1 (ARG1, ab269541; Abcam), interferon gamma (IFNɣ, ab282874; Abcam), and CD163 (ab272204; Abcam). Immunocytochemistry for cleaved caspase 3 and caspase 9 was done using rabbit anti-mouse antibodies (Abcam).

The umbilical cord tissues were homogenized (100 μL), placed in the reaction tube, lysed using 1 mL of protease-containing cell lysate for 30 min at 4 °C, shaken once every 10 min at 4 °C, and centrifuged at 6000 r/minute for 20 min. After the lipid layer was discarded, the supernatant was taken as protein extract. Protein concentration was measured utilizing a bicinchoninic acid kit (20201ES76, Yeasen Biotechnology, Shanghai, China). Proteins were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by transferring onto the polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk at room temperature for 1 h and incubated with primary rabbit anti-mouse antibodies (Abcam Inc., Cambridge, UK) to HDAC2 (ab32117, 1:2000), p53 (ab131442, 1:1000), p53 Ac (acetyl K373) (ab75754, 1:1000), PUMA (ab33906, 1:2000), Ki67 (ab92742, 1:5000), PCNA (ab152112, 1:1500), MMP-2 (ab37150, 1:2000), MMP-9 (ab73734, 1:2000), Bax (ab32503, 1:2000), Bcl-2 (ab59348, 1:1000), and GAPDH (ab8245, 1:5000) overnight at 4 °C. The next day, the membrane was reacted with horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) (ab6721, 1:5000) at room temperature for 1 h. The membrane was developed by ECL imager. Quantity One software was used to quantify the protein-expression level with GAPDH as an internal control.

The cells were immersed in PBS and seeded in glass slides at a concentration of 1 × 10⁴/ml. The slides were fixed in 4% paraformaldehyde for 15 minutes, then immersed in PBS 3 times for 3 minutes each time, and subsequently in 0.5% Triton X‐100 in PBS at room temperature for 20 minutes. Next, the cells were blocked with 5% skim milk powder in PBST for 30 minutes at room temperature, and the following diluted primary antibodies were added: anti‐Lamin B, BOSTER, China; anti‐β‐actin, Abcam, USA; anti‐Lamin A, Abcam, USA. The antibodies used were rabbit anti‐human and diluted at a concentration of 1:200. The cells were incubated overnight at 4°C and subsequently with the fluorescent secondary antibody (1:100, mouse anti‐rabbit antibody, Abcam, USA) for 1 hour at room temperature. Finally, the slides were washed in PBST 3 times for 3 minutes each and observed under a fluorescence microscope.

RIPA lysis buffer (plus PMSF) was used to lyse the cells to extract the total proteins, which were separated by a polyacrylamide gel electrophoresis and transferred into the PVDF membrane. After membrane blocking, the corresponding primary antibodies (1:1000, rabbit anti‐human, anti‐Lamin A/Lamin C/β‐actin, Abcam, USA, and anti‐P16/CDK1/MMP2/MMP9, BOSTER, China) were added to the membrane that was incubated overnight at 4°C. Next, the secondary antibody (1:8000, mouse anti‐rabbit antibody, Abcam, USA) was added to the membrane that was incubated at room temperature for 1 hour. Finally, the bands were imaged using Gel imaging system.