To check for levels of p53 expression and cleaved PARP (indicator of apoptosis), the tissues were sectioned to 14-μm thickness, fixed with 4% paraformaldehyde, and blocked with blocking buffer for 1 hr. This was followed by treating with anti-p53 (Sigma; P 8999) or anti-cleaved PARP (Thermo Fisher Scientific; 44-698G) antibodies overnight in blocking buffer. On subsequent day, the sections were treated with fluorescent secondary antibodies. Sections were imaged on a Zeiss LSM 700-405 confocal microscope.
To check for levels of TNF-α (pro-inflammatory cytokine), ki67 (cell proliferation), CD68 (activated microglia), and cleaved PARP (apoptosis), the tissues were sectioned in 4- to 5-mm blocks and submitted as individual cassettes for paraffin embedding at Emory Winship Pathology Core Lab. Tissues from paraffin-embedded blocks were sectioned at 5-μm thickness. IHC was performed using DAB chromogenic kit (Wako) following the manufacturer’s protocol. Antibodies used were anti-TNF-α (Boster Biologics; PA 1079), anti-p53 (Sigma; P8999), anti-cleaved PARP (Thermo Fisher Scientific; 44-698G), anti-ki67 (Thermo Fisher Scientific; MA5-15690), and anti-CD68 (AbD Serotec; MCA 341R). Whole-slide scanning was done using Hamamatsu’s Nanozoomer 2.0 HT.
To check for levels of TNF-α (pro-inflammatory cytokine), ki67 (cell proliferation), CD68 (activated microglia), and cleaved PARP (apoptosis), the tissues were sectioned in 4- to 5-mm blocks and submitted as individual cassettes for paraffin embedding at Emory Winship Pathology Core Lab. Tissues from paraffin-embedded blocks were sectioned at 5-μm thickness. IHC was performed using DAB chromogenic kit (Wako) following the manufacturer’s protocol. Antibodies used were anti-TNF-α (Boster Biologics; PA 1079), anti-p53 (Sigma; P8999), anti-cleaved PARP (Thermo Fisher Scientific; 44-698G), anti-ki67 (Thermo Fisher Scientific; MA5-15690), and anti-CD68 (AbD Serotec; MCA 341R). Whole-slide scanning was done using Hamamatsu’s Nanozoomer 2.0 HT.