Ab109508

CRC cells and macrophages were lysed by the RIPA solution to extract the protein (Thermo Fisher Scientific, Waltham, MA, USA), then the protein concentration was measured with the BCA Protein Assays Kit (Beyotime, Shanghai, China). The proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane, which was then blocked for 2 h at room temperature, and subsequently incubated with primary antibodies, including anti-GAPDH (5174, 1:1000, Cell Signal Technology, Danvers, MA, USA), anti-TGF-β1 (ab215715, 1:1000, Abcam, Cambridge, UK), and anti-CST3 (ab109508, 1:1000, Abcam, UK) overnight at 4 °C. Next, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (7076, 1:1000, Cell Signal Technology, Danvers, MA, USA) for 2 h at room temperature. Blots were visualized by enhanced chemiluminescence on a Tanon 5200 system (Tanon, Shanghai, China).

Following SDS-PAGE using 12% gels, proteins were transferred to a nitrocellulose membrane using standard molecular biology procedures. Primary antibodies used were against cystatin C (Abcam, Cambridge, UK; ab109508), GAPDH (Abcam, Cambridge, UK; ab8245), CYB5B (Thermo Scientific, Waltham, MA, USA; PA5-52482), TOM20 (Cell Signaling Technology, Danvers, MA, USA; 42406), TSPO (Abcam, Cambridge, UK; ab109497), IRAP (also known as leucyl-cystinyl aminopeptidase; Cell Signaling Technology, Danvers, MA, USA; 6918), and α-tubulin (Abcam, Cambridge, UK; ab4074). After blocking in 5% milk in TBS supplemented with 0.1% Tween-20 (TBST) and probing with primary antibody, membranes were washed three times with TBST and incubated with secondary antibody conjugated to HRP (Sigma, St. Louis, MO, USA; A0545 or A9044), and then washed for an additional three times with TBST and developed using either Radiance Plus chemiluminescent substrate (Azure Biosystems, Dublin, CA, USA) or SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo scientific, Waltham, MA, USA). Blots were imaged using a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA). Band intensity was assessed via densitometry and normalized against indicated housekeeping protein, and was used as a semi-quantitative estimate of protein concentrations.

Paraffin sections were rehydrated and antigen retrieval was performed using citrate buffer (pH 6). The following antibodies were used: rabbit anti pS6 (5364S, Cell Signaling), rabbit anti Cystatin-C (ab109508, Abcam), rabbit anti collagen III (ab7778, Abcam), rabbit anti TNFα (ab6671, Abcam), goat anti-GFP (ab6673, Abcam), mouse anti SMA (ab7817, Abcam), rabbit anti-ZO1 (ab216880, Abcam), rabbit anti E-cadherin (ab15148, Abcam), rabbit anti-Ki67 1:200 (Thermo Scientific), rabbit anti-pAKT S473 (4060S, Cell Signaling), rabbit anti HIF-1 alpha (ab82832, Abcam). Cell nuclei were visualized with DAPI staining. Secondary antibodies were all from Jackson ImmunoResearch Laboratories. Digital images were obtained with a Nikon A1R confocal microscope. Fluorescence was quantified using the ImageJ software (NIH, Bethesda, MD).

After being treated, different groups of samples were washed with cold PBS and incubated with RIPA buffer containing protease inhibitor cocktail. Lysates were centrifuged at 12,000 g for 10 min. Protein concentration was quantified by BCA Protein Assay. The protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% fat-free milk in TBST buffer for 90 min and incubated overnight at 4°C with primary antibodies. The primary antibodies were showed as follows: Cys-C (1:1000, Abcam, #ab109508), β-catenin (1:1000, Abcam, #ab16051),α-actin (1:1000, Abcam, #ab179467). After washing with TBST, the membranes were incubated with horseradish peroxidase-coupled secondary antibodies for 2 h at room temperature. The secondary antibody was α-actin (1:1000, Abcam, #ab179467). After all procedures, the images were taken with enhanced chemiluminescence (ECL) reagent and exposure in the dark areas. All data were reproduced three times and analyzed by ImageJ software.

LGs were fixed, sectioned, and prepared for immunofluorescence as described previously^{54 (link)}. Sections were incubated with primary rabbit anti-Cys C antibody (ab109508, abcam) diluted 1:100, followed by anti-rabbit AlexaFluor488-conjugated secondary antibody (A21206, Molecular Probes, Eugene, OR) diluted 1:200. Samples were mounted with ProLong anti-fade mounting medium (Molecular Probes) and imaged by confocal fluorescence microscopy using an LSM 510 Meta NLO system (Carl Zeiss, Oberkochen, Germany).

After different treatments, tissues were immersed in 4% paraformaldehyde for 4 h and transferred to 70% ethanol. Then, tissue was placed in cassettes, dehydrated through a serial alcohol gradient, and embedded in paraffin wax blocks. Finally, the tissue was stained with hematoxylin and eosin. Histopathological examination was performed in infarcted tissue on formalin-fixed, paraffin-embedded 5 to 6 μm sections stained with hematoxylin and eosin using the standard methods and examined via light microscopy. Antibodies used in this study were as follows: Cys-C (1:400, Abcam, #ab109508), a TUNEL assay was applied to observe the necroptosis according to the manufacturer’s protocol (R&D systems).

Four-micrometer sections of methyl-Carnoy fixed, paraffin-embedded tissue sections were immunostained as described previously [15] (link). Cystatin C antibody was obtained from Abcam (ab109508; Abcam, Cambridge, UK). Negative controls for the immunohistochemical procedures consisted of substitution of the primary antibody with nonspecific IgG.