Activated caspase 3

Mandibles from 2- to 3-month-old heterozygous and homozygous Enam^p.S55I mutant and wild-type mice (n = 4 in each category) were dissected following cervical dislocation and fixed in 4% paraformaldehyde in PBS, pH 7.4, at room temperature for 48 h. Following fixation, the mandibles were decalcified in 0.5 M EDTA, dehydrated through a graded ethanol series, cleared in chloroform, embedded as hemi-mandibles in paraffin wax, sectioned and stained with haematoxylin and eosin. Sections were examined using a DMRB microscope (Leica) with SpotTM digital camera and associated software (RTKE/SE Diagnostic Instruments Inc.). Immunofluorescence analysis was performed on mandibles prepared as above using antibodies raised against activated caspase 3 (Abcam, Cambridge, UK), amelogenin FL191, (Santa Cruz Biotechnology, Santa Cruz, CA), ameloblastin (C17, Santa Cruz Biotechnology, Santa Cruz, CA), enamelin, phosphohistone H3 (Abcam, Cambridge, UK), keratin 14 (Abcam, Cambridge, UK) and GRP-78 (Abcam, Cambridge, UK).
Primary antibodies were detected using biotinylated secondary antibodies (Vector laboratories) followed by Cy-3-conjugated streptavidin (Sigma, Poole, UK) or an AlexaFluor488-conjugated secondary antibody (Abcam, Cambridge, UK) and the sections were mounted in fluorescence mountant containing DAPI (Vector laboratories).

Thirty days after the last measurement of tumor volume in prophylactic immunotherapy, mice were sacrificed and paraffin-embedded tumor tissue sections were used for the examination of PCNA, activated-caspase-3 (Abcam, Cambridge, UK) and tunnel (Promega, Madison, WI). Sections were scored under light microscopy (X200) by three independent pathologists, who analyzed three different fields per section.

The whole-cell lysates were generated using the Whole Cell Lysis Assay Kit (KeyGEN, China) according to the manufacturer’s protocol. The protein concentration was detected by the BCA method (Thermo Fisher Scientific, USA). β-Actin (1: 1000, Abcam, UK) was used as an internal control. Primary antibodies included ISL1 (0.5 μg/mL, Developmental Studies Hybridoma Bank, USA), GATA3 (1:1000, Invitrogen, USA), E-cadherin (1:10,000; Abcam, UK), N-cadherin (1:5000; Abcam, UK), vimentin (1:1000; Abcam, UK), snail + Slug (1 μg/mL; Abcam, UK), Bcl2 (1:1000; Abcam, UK), Bax (1:1000; Abcam, UK), Activated Caspase3 (1:1000; Abcam, UK), AKT (1:1000, Cell Signaling Technology, USA), p-AKT (1:1000, Cell Signaling Technology, USA), and AURKA (1:1000; Abcam, UK). Secondary antibody included goat anti-rabbit antibody (1:10,000, Jacson ImmunoResearch, USA) or goat anti-mouse antibody (1:10,000, Jacson ImmunoResearch, USA). The ECL chemiluminescence detection system (Bio-Rad, USA) was used for signal detection.

For cryosection analysis, heads or enucleated eyes were fixed in 4% (w/v) PFA in PBS for up to 2 h on ice, equilibrated overnight at 4°C in 30% (w/v) sucrose in PBS and embedded in OCT compound (Sakura Finetek). For flat-mount analysis, retinae were dissected out from enucleated eyes and individually placed in separate wells of a 24-well plate and fixed in 4% (w/v) PFA in PBS for 2 h on ice. Immunohistochemistry was performed on cryosections (10-20 µm) or flat-mount retinae as previously described (Cantrup et al., 2012 (link); Ma et al., 2007 (link)). The following antibodies and dilutions were used: pS6 (S235/236) (Cell Signaling Technology, 1:100), Brn3 (Santa Cruz Biotechnology, 1:50), activated caspase3 (Abcam, 1:1000), NF (SMI31) (Covance, 1:1000), GFAP (Chemicon, 1:400), Pax6 (DSHB, 1:100), ChAT (Millipore, 1:100), Calretinin (Swant, 1:1000), PKCα (Santa Cruz Biotechnology, 1:100), Calbindin-D (Sigma-Aldrich, 1:100) and p27^Kip1 (BD Biosciences, 1:750).

Ipsilateral cortices were collected 24 h after ischemia and processed for western blotting as previously described [19 ]. Antibodies against the following proteins were used at 1:1000 dilution: activated caspase-3 and Bcl-2-associated X protein (Bax) (both from Abcam, Cambridge, UK), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands were detected using horseradish peroxidase-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology) and by enhanced chemiluminescence.