Cleaved caspase 3 5a1e

Tissue samples from xenograft tumors (including xenograft subcutaneous tumor model, xenograft metastatic model and PDX model) were fixed in 4% formaldehyde for 24 hours, processed to the paraffin, and sectioned. For immunohistochemistry, 5 μm sections were deparaffinized in xylene and hydrated in alcohol. Endogenous peroxidase activity was then blocked by incubation in 3% hydrogen peroxide-methanol for 10 minutes. For the antigen recovery, the section was heated in a citrate buffer (10 mM Sodium Citrate, pH 6.0) for 15 minutes. After blocking, the primary antibodies against caspase-3(1:1000) (Lot: #9662, Cell Signaling), cleaved caspase-3 (5A1E) (1:400) (Lot: #9664, Cell Signaling) was applied at 4℃ overnight. EnVision+ TM (Dako) was used as the secondary antibody. Antibody binding was visualized by a standard streptavidin immunoperoxidase reaction. These sections were counterstained with hematoxylin.
The score of immunohistochemistry was graded by histochemistry score (H-Score) system. Evaluation was carried out independently by two experienced pathologists. The H-score system was the combination of proportional and intensity of positive tumor cells. H score = ΣPi(i+1). Pi, the ratio of the positive staining cell in all the tumor cells; i, the staining intensity.

Tissue samples from xenograft tumors (including xenograft subcutaneous tumor model, xenograft metastatic model and PDX model) were fixed in 4% formaldehyde for 24 hours, processed to the paraffin, and sectioned. For immunohistochemistry, 5 μm sections were deparaffinized in xylene and hydrated in alcohol. Endogenous peroxidase activity was then blocked by incubation in 3% hydrogen peroxide-methanol for 10 minutes. For the antigen recovery, the section was heated in a citrate buffer (10 mM Sodium Citrate, pH 6.0) for 15 minutes. After blocking, the primary antibodies against caspase-3(1:1000) (Lot: #9662, Cell Signaling), cleaved caspase-3 (5A1E) (1:400) (Lot: #9664, Cell Signaling) was applied at 4℃ overnight. EnVision+ TM (Dako) was used as the secondary antibody. Antibody binding was visualized by a standard streptavidin immunoperoxidase reaction. These sections were counterstained with hematoxylin.
The score of immunohistochemistry was graded by histochemistry score (H-Score) system. Evaluation was carried out independently by two experienced pathologists. The H-score system was the combination of proportional and intensity of positive tumor cells. H score = ΣPi(i+1). Pi, the ratio of the positive staining cell in all the tumor cells; i, the staining intensity.

Anti-pSer40/41MCM2 antibody was raised by GenScript (GenScript USA Inc) using a CAPLT(p)S(p)SPGR peptide and further affinity purified. Cleaved PARP (19F4) and cleaved Caspase 3 (5A1E) antibodies were from Cell Signalling Technology; CDC7 (clone SMP171) and β-tubulin (ab6046) antibodies were from Abcam; β-Actin antibody (Cat No. A00702) was from GenScript; MCM2 antibody (Cat No. MCA1859) was from AbD Serotec; pSer41MCM2 antibody (Cat no. A300-117A-1) was from Bethyl Laboratories Inc; γ-H2AX antibody, recognizing pSer139-H2AX (Cat No. 05-636) was from Millipore; pSer108MCM2 antibody was previously described [9] (link). Secondary antibodies labeled with infrared fluorophores (800CW anti-rabbit Cat No. 926-32211, 800CW anti-mouse Cat no. 926-32210, 680LT anti-rabbit Cat No. 926-68021 and anti-mouse Cat no. 926-68020) were obtained from Li-COR Biosciences Ltd, secondary antibodies labeled with TRITC fluorophore were obtained from Jackson ImmunoResearch. Odyssey Infrared Imaging Systems were from Li-COR Biosciences Ltd.

After deparaffinization and rehydration, antigen retrieval was performed by boiling in sodium citrate buffer using a microwave. Endogenous H₂O₂ was inactivated by incubating with 0.3% H₂O₂ for 30 min. Nonspecific binding was blocked by incubating with normal horse serum for 20 min. Sections were then incubated with 1:1,000 4-HNE (ab46545, Abcam, Cambridge, UK) or cleaved caspase-3 (5A1E, Cell Signaling Technology, MA, USA) primary antibody at 4 °C overnight, followed by incubation with secondary antibody at 37 °C for 30 min. After washing, sections were incubated with ABC reagent (VECTASTAIN® ABC kit) for 30 min, soaked in 1% diaminobenzidine staining reagent for 5 min, and counterstained with hematoxylin for 5 min. Finally, sections were dehydrated, cleared, mounted, and visualized under a light microscope.

IHC was done as previously described⁶⁰ . Details are shown in Supplemental information. Primary antibodies used were: C-pro/α1(I) (600-401-D19, RL, Rockland, 1:200);: C-pro/α1(I) (LF42, Kerafast, 1:4000); VIM (ab92547, Abcam, 1:2000); lamin A/C (ab108595, Abcam, 1:2500); human ColI/α1(I) (AF6220, R&D systems, 1:75); human ColI/α2(I) (A5786, Abclonal, 1:500); mouse ColI (AB765P, Millepore, 1:100); BMP1 (ab118520, Abcam, 1:250); Ki67 (SP6, VALENT, 1:50); and Cleaved caspase 3 (5A1E, Cell Signaling, 1:800).