Alexa 546 goat anti rabbit secondary antibody

To compare the effects of drug treatment on cell proliferation, we labeled Ki67, a protein expressed by cells in all phases of cell cycle except G0 and early G1. For this procedure, slides were first washed in PBS, then bathed in 10 mM sodium citrate (pH 6.0) at 95⁰ C for 15 min to induce antigen retrieval before cooling to room temperature. After washes in PBS, the tissues were blocked with a mix of 5% NGS, 1% BSA and 0.3% Triton-X 100 for 1 hr at room temperature. The rabbit anti-Ki67 primary antibody (1:200 dilution; catalog # SP6, ThermoFisher Scientific; RRID: AB-2341197) was added to the tissues, then covered with hybristrips for incubation overnight at 4⁰ C. The following day sections were washed in PBS before they were incubated for two hrs in Alexa 546 goat-anti-rabbit secondary antibody (1:1000 dilution, Invitrogen, ThermoFisher Scientific, Waltham, MA, USA RRID: AB_2534093) at room temperature. Sections were then washed in PB before bathed in Sytox green (1:30,000, catalog # S7020, Molecular Probes, Eugene, OR, USA) to double label nuclei. After additional washes, cover slips were mounted using Fluoromount G (#0100–001, Southern Biotech, Burmingham, AL, USA).

Since TUNEL can label fragmented DNA from either necrotic activity or apoptosis, we looked at cleaved caspase-3 as a marker that detects cells in a specific phase of apoptosis. The role of caspase-3 in apoptosis is to cleave and activate caspases-6, 7 and 9 prior to the apoptotic cell breaking down before removal [57 (link)]. Cleaved caspase-3 assays were conducted on tissues at 0, 2, 4, 6, 8, 12, 18, 24, 36 and 48 hrs post injection. The protocol was adapted from French et al. [58 (link)]. Briefly, the slides were exposed to 200ug/ml proteinase K in PBS for 15 min at 37⁰ C for antigen retrieval. The sections were exposed to rabbit anti-cleaved caspase-3 primary antibody (Cat# 9661S, Cell Signaling, Danvers, MA, USA; RRID: AB-2341188) at a concentration of 1:100 overnight at 4⁰ C. After washing in PBS, the sections were incubated in Alexa 546 goat-anti-rabbit secondary antibody (1:1000 dilution, Cat# A-11010, Invitrogen, ThermoFisher Scientific, Waltham, MA, USA; RRID: AB-2534093) for 2 hrs at room temperature. The tissues were then counter-reacted with Sytox green.