Anti cleaved caspase 3 5a1e

Sections from large intestines were collected from 6-week-old mice. Paraffin sections (5 μm) were deparaffinized in xylene and rehydrated in an ethanol gradient. Antigen retrieval was performed in Rodent Decloaker using a Decloaking Chamber (Biocare Medical, Walnut Creek, CA). Endogenous peroxidase activity was quenched by incubation in 3% H₂O₂ for 10 min, and slides were blocked with Dako Protein Block (DakoCytomation Inc., Mississauga, Ontario, Canada) for 30 min at room temperature. The slides were incubated with primary antibody for 1 h at room temperature. The following polyclonal antibodies were used: anti-cleaved caspase-3 (5A1E, 1:800) and Ki-67 (12202, 1:400) (Cell Signaling Technology, Beverly, MA). After washing, slides were incubated with prediluted biotinylated goat anti-rabbit IgGs (Biocare Medical) for 30 min and then treated with 3,3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories, SK-4100). Samples were mounted with aqueous mounting medium (Dako, S3025), and images were taken on a Nikon microscope using NIS-Elements software.

Murine ovarian cancer or subcutaneous cancer tissue specimens were fixed in paraformaldehyde and then embedded in paraffin. After sectioning (3 μm thick), paraffin-embedded cancer tissues were used for immunostaining with anti-pSTAT3 (D3A7; Cell Signaling Technology), anti-Iba-1 (WAKO), anti-F4/80 (clone CI:A3-1, Serotec, Ltd., Oxford, UK), anti-CD163 (rabbit polyclonal, Cosmo Bio, Tokyo, Japan), anti-Ki67 (DAKO, Glostrup, Denmark), anti-cleaved caspase-3 (5A1E; Cell Signaling Technology, Beverly, MA, USA) and anti-CD3 (Abcam, Cambridge, UK) antibodies.
Murine tissues were also embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA, USA), snap-frozen in liquid nitrogen and stored at −80 °C. After sectioning (7 μm thick), the tissues were fixed with cold acetone and treated with the following primary antibodies: anti-CD4 (GK1.5; ATCC, Manassas, VA, USA) and anti-CD8 (53–6.72; ATCC, Manassas, VA, USA). The sections were subsequently treated with an HRP-conjugated secondary antibody (Nichirei, Tokyo, Japan), and the reactions were visualized with diaminobenzidine. The number of immunopositive cells was counted using the BZ-9000 analysis software programme (Keyence, Osaka, Japan).

Anti‐Gli1 (cat# 2553), anticleaved caspase‐3 (5A1E, cat# 9664), anti‐STAT3 (cat# 9139), and anti‐p‐STAT3 (Tyr705) (cat# 4113) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐p21 (F‐5, sc‐6246) and anti‐Sox2 (E‐4, sc‐365823) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Western blot of whole‐cell lysates harvested in lysis buffer was performed with primary antibodies, and then, goat anti‐mouse or rabbit horseradish peroxidase secondary antibodies were used and the immunoreactive bands were detected by using SuperSignal West Pico Chemiluminescent substrate, or SuperSignal West Dura Extended Duration substrate (Thermo Fisher Scientific, Waltham, MA, USA). All experiments for western blot were performed at least three times except where indicated in the figure legends. Western blot protein bands were quantified by using imagej, the ratio of each protein band relative to the lane's loading control was calculated, and the final relative values are the ratio of net protein band to net loading control. The ratio for each control cell was normalized as 1, then calculated the relative fold change for each lane relative to the control in each cell line.