Immunohistochemistry for Apoptosis and Senescence

For cleaved caspase-3 or Ki-67 immunohistochemistry (IHC), spleens and lymph nodes from lymphoma-bearing mice were fixed in 10% formalin and then embedded in paraffin. Sections were stained for cleaved caspase-3 using the SignalStain Apoptosis (Cleaved Caspase-3) IHC Detection Kit (Cell Signaling Technology) according to the manufacturer’s protocol and counterstained with haematoxylin. Ki-67 IHC was performed using D3B5 Rabbit mAb (Cell Signaling Technology). To assess senescence, we followed the protocol of Post et al.^{36 (link)} Briefly, lymph nodes from lymphoma-bearing mice were fixed for 2 h in 4% paraformaldehyde, and then incubated for at least 6 h in 10% sucrose/Hanks’ Balanced Salt Solution (HBSS), followed by 15% sucrose/HBSS, then 20% sucrose/HBSS, before snap freezing in Tissue-Tek O.C.T. Compound (Sakura Finetek, Torrance, CA, USA). Cryosections were stained for SA-β-galactosidase as previously described,^{9 (link)} and counterstained with nuclear fast red. Complete slides were scanned on an Aperio Digital Pathology Slide Scanner and regions to be analysed were extracted using ImageScope software (Leica Biosystems, Wetzlar, Germany). Cleaved caspase-3 or β-galactosidase-positive cells were quantified using Fiji software (ImageJ, NIH, Bethesda, MD, USA).^{39 (link)}