Image pro plus software 6

Mouse tumors were isolated and fixed with 4% paraformaldehyde, followed by preparing paraffin sections, deparaffinization, antigen retrieval, blocking, and incubating tumor tissues with antibodies against mouse CD31 and F4/80 (CST, United States) overnight at 4°C. The following steps were performed as informed in the rabbit two-step detection kit (ZSGB-BIO, Beijing, China). Briefly, after the primary antibody was bound to the antigen in the tissue, HRP-conjugated antibodies were added separately, using DAB as the chromogenic reagent. The images were collected using an optical microscope (Zeiss, Oberkochen, Germany), and the data were analyzed using Image-Pro Plus software 6.0 (RRID:SCR_016879, Media Cybernetics, United States).

Cells were collected and resuspended in RIPA lysis buffer for 60 min incubated on ice. After centrifuging for 20 min at 12000g and 4°C, the supernatant was collected. Protein concentrations were determined by using Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). 60 μg of protein was separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was rinsed with TBST and incubated in 5% nonfat milk diluted with TBST at 37°C for 2 h. The membrane was then incubated with mouse anti-Bak (1:1000 dilution, Cell signaling technology), mouse anti-c-PARP (1:1000 dilution, Cell signaling technology), and mouse anti-GAPDH primary antibodies (1:3000 dilution, Santa Cruz, Dallas, Texas, USA) respectively overnight at 4°C. After washing with TBST 3 times for 15 min each time, the membrane was incubated with the appropriate secondary antibodies for 1 hour at 37°C. The membranes were then washed with TBST 4 times for 10 min each time and visualized the bands by using an ECL kit (Millipore, Billerica, MA) in a Gel Doc™ XR+ and ChemiDoc™ XRS+ System (BioRad, Hercules, CA). Image-Pro plus software 6.0 (Media Cybernetics, Rockville, MD, USA) was used to analyze the relative protein expression which was normalized to GAPDH.

Tissues and cells were lysed in cold radioimmunoprecipitation assay lysis buffer. The proteins were separated with 10% SDS-PAGE, and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated overnight with phosphate-buffered saline containing 5% milk at 4°C. Following incubation, the PVDF membrane was incubated with monoclonal mouse anti-human Spry2 (1:200; ab60719) and monoclonal mouse anti-human GAPDH primary antibodies (1:200; ab125247; Abcam, Cambridge, UK) at room temperature for 3 h, and then incubated with polyclonal rabbit anti-mouse secondary antibodies (1:5,000; ab175743; Abcam) at room temperature for 1 h. An electrochemiluminescence kit (Pierce Chemical, Rockford, IL, USA) was then used to perform chemiluminescent detection. The relative protein expression was analyzed by Image-Pro plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and was presented as the density ratio versus GAPDH.

Paraffin embedded tissue blocks were cut at a thickness of 5 μm and stained with hematoxylin and eosin (HE) for pathological assessment. Fixed sections were dewaxed and hydrated before antigens recovery by repeated cooling and heating. Sections were incubated in the indicated primary and secondary antibodies for detection of positive particles by diaminobenzidine (DAB). Color development was observed using a digital camera (Olympus, Tokyo, Japan) and calculated using Image-Pro Plus software 6.0 (Media Cybernetics, Silver Spring, MD, USA).

Paraffin sections were completely melted in an electrothermal constant temperature oven and dewaxed in xylene. After dehydration with ethanol, endogenous peroxidase activity was blocked by 3% hydrogen peroxide solution. Citrate buffer (pH 6.0) was used to recover the antigens by cooling and heating repeatedly. The sections were then blocked with 5% bovine serum albumin (BSA) and incubated overnight with primary antibodies including TNF-α (1:100), IFN-γ (1:100), Bid (1:50), Caspase 8 (1:100), Caspase 9 (1: 50), TRADD (1:100), p-FADD (1:50), Bcl-2 (1:50), p-JAK2 (1:100), p-STAT1 (1:100). On the second day, the slices were washed three times and incubated with secondary antibodies. Diaminobenzidine (DAB) was applied to show positive particles. Color development was observed using a digital camera (Olympus) and calculated using Image-Pro Plus software 6.0 (Media Cybernetics, Silver Spring, MD, USA).