Abc reagent

TUNEL assays were done as described (Arama and Steller, 2006 (link)) with some deviations. DIG-labelled nucleotides were detected with a sheep anti-DIG antibody (Roche) and a donkey biotinylated anti-sheep secondary antibody (Jackson ImmunoResearch). For signal amplification, embryos were incubated for 45′ with ABC reagents (Vector Laboratories), followed by a 5′ incubation with TSA Flourescein reagents (Perkin Elmer) diluted 1:50. Embryos were mounted in ProlongGold (Life Technologies).

Fetal gonads were dissected in phosphate buffered saline (PBS), fixed in 4% paraformaldehyde overnight at 4°C, embedded in paraffin and sectioned. Slides were incubated with anti-GCNA IgM (courtesy of G. Enders, undiluted supernatant), anti-STRA8 (Abcam. 1∶100), and anti-phosphoH2A.X (Upstate Cell Signaling Solutions, 1∶250 dilution). Colorimetric staining was performed using ABC reagents (Vector Laboratories) and developed with DAB peroxidase substrate (Vector Laboratories).
Sections were mounted in Vectashield Medium with DAPI (Vector Laboratories), and fluorescent staining was obtained using Texas-Red or FITC-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, 1∶500 dilution).

Western blotting was performed as described in our previous report.^{(9 (link))} A549 cells were seeded in a 6-cm-diameter dish (1.0 × 10⁶ cells/dish). After the treatment, cells were washed twice with ice-cold PBS and lysed using 150 µl of the lysis buffer. Aliquots of the prepared samples were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was sequentially incubated with each primary antibody (1:3,000), biotin-conjugated secondary antibody (1:3,000), and ABC reagents (Vector Laboratories, Burlingame, CA) (1:5,000). Finally, proteins were detected using Super-Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) or ImmunoStar LD (Wako Pure Chemical), and imaged using ChemDoc Touch (Bio-Rad, Hercules, CA).

The substantia nigra regions of the brains of PD and control mice were collected and fixed in 4% paraformaldehyde. Following serial coronal sectioning (30-μm sections), the brain tissues were blocked with 5% goat or horse serum/PBS plus 0.2% Triton X-100. Then, the sections were incubated with an anti-TH antibody (Novus Biologicals) or an anti-Iba-1 antibody (Wako) before being incubated with a biotin-conjugated anti-rabbit antibody (Vector Labs). After 2 h, the sections were washed with PBS three times, incubated with ABC reagents (Vector Labs) and then developed using Sigma Fast 3,3′-Diaminobenzidine (DAB) Peroxidase Substrate (Sigma-Aldrich, St. Louis, MO, USA). Nissl (0.99% thionin) was used to counterstain the sections.

The other serial sections underwent pretreatment with trypsin (Sigma-Aldrich) and 2 N hydrochloric acid for antigen retrieval. Endogenous peroxidase activity was removed using 0.3% hydrogen peroxide and a non-specific binding protein was blocked with normal horse serum for 1 h. Then, the sections were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal antibodies for TNFα (#sc-52746, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200) and BrdU (#ab8152, Abcam, Cambridge, UK; 1:100), and rabbit polyclonal antibodies for COX-2 (#160126, Cayman; 1:200) and cleaved PARP (#9545, Cell Signaling Technology Inc., Danvers, MA, USA; 1:100). The next day, the sections were incubated with horse biotinylated secondary anti-mouse IgG or anti-rabbit IgG antibody for 1h, followed by ABC reagents (#PK-6200, Vector Lab., Burlingame, CA, USA) for 30 min. Immunoreactivity was visualized using a peroxidase substrate kit (Vector Lab.) and counterstained with hematoxylin. The staining process was conducted in a humidity chamber, and sections were rinsed with PBS three times between each step. Immunostains omitting primary antibodies were used as the negative controls. Cells occupying immunoreactive regions over 20% were counted by a histopathologist blinded to the experimental groups.

Mouse kidney samples were fixed in 10% neutraformaline, embedded in paraffin. 3 μm sections were used for Masson, PAS and Sirius red staining. For immunohistochemical staining, paraffin-embedded kidney sections were deparaffinized, hydrated, antigen-retrieved, and endogenous peroxidase activity was quenched by 3% H₂O₂. Sections were then blocked with 10% normal donkey serum, followed by incubation with anti-β-catenin (cat: 61054, BD Biosciences Pharmingen, San Diego, CA), anti-p-4E BP (T37/46) (cat: 2855, Cell Signaling Technology, USA) and anti-p-Smad3 (S423/S425) (cat: ab52903, Abcam, Cambridge, UK) overnight at 4 °C. After incubation with secondary antibody for 1 hour at room temperature (RT), sections were incubated with ABC reagents for 1 hour at room temperature before subjected to substrate 3-amino-9-ethylcarbazole or DAB staining (Vector Laboratories, Burlingame, CA). Slides were viewed with a Nikon Eclipse 80i microscope equipped with a digital camera (DS-Ri1, Nikon, Shanghai, China).