Anti mouse irdye 800cw

Western blot analysis was performed as previously described (Salmon et al., 2012a (link)) with modifications for detection of CARP1. Briefly, lysates of 3-5×10⁶ trypanosomes were separated on 10% polyacrylamide gels, transferred to a PVDF membrane via semi-dry blotting and blocked with Kem-En-Tec synthetic blocking buffer for 1 h at room temperature. The blots were incubated with primary antibodies (rabbit anti-CARP1, 1:1000; mouse anti-PFR-A/C [(Kohl et al., 1999 (link)), 1:2000; PFR, paraflagellar rod protein)] overnight at 4°C, followed by secondary antibody (IRDye680LT anti-rabbit and IRDye800CW anti-mouse, LI-COR, both 1:5000) detection for 1.5 h at room temperature. For detection of His-tagged proteins expressed in E. coli, proteins were blotted onto PVDF membranes and blocked with 5% milk for 1 h at room temperature, followed by incubation with mouse anti-His (1:1000, BioRad MCA1396GA) overnight at 4°C and secondary antibody detection with IRDye800CW anti-mouse (1:5000, LI-COR).

Proteins were extracted using RIPA Lysis buffer containing Phosphatase Inhibitor Cocktail (Sigma Aldrich, Sydney, Australia) according to the manufacturer’s instructions. Protein concentration was quantified using a BCA Protein Determination Kit (Pierce) as per the manufacturer’s instructions and absorbance read at 562 nm on a PerkinElmer VICTOR X Multilabel Plate Reader. Western blots were performed as previously described [19 (link)], utilizing rabbit antibodies against phospho-STAT3 (Tyr 705), total-STAT3, BCL2 and ICAM-1 (Cell Signaling, Beverly, MA, USA), and a mouse monoclonal antibody against GAPDH (Millipore Technologies, Sydney, Australia) at 1:1000 dilution, followed by anti-rabbit IRDye™ 680 CW and anti-mouse IRDye™ 800 CW (LI-COR Biosciences, Nebraska, USA) at 1:10000 dilution in 5% (w/v) BSA in TBS containing 0.1% Tween. Images were obtained using an Odyssey Imaging System (LI-COR Biosciences) and analysed with ImageJ (NIH).

Forty-eight male Sprague-Dawley (SD) rats weighing between 250 and 300 grams were obtained from the Animal Experimental Centre of Guangxi Medical University. The animal handling protocol was in accordance with the Regulations of Laboratory Animal Care [3 ]. The rats were housed singly in polycarbonate cages at 24°C in a humidity-controlled environment with a 12-hour day/night cycle during the preoperative and postoperative periods. The rats were allowed free access to water and rat chow.
Lyophilized natural hirudin powder (Patent number ZL03113566.8, Lot number KK-001) was provided by Nanning JinXueHuang Bioengineering Co. Ltd. (Guangxi, China). Anti-Pp38-MAPK rabbit antibody (cat. #4511), anti-p38-MAPK rabbit antibody (cat. #8690), and anti-GAPDH rabbit antibody (cat. #5174) were purchased from Cell Signalling Technology (Beverly, MA, USA). Anti-ICAM-1 mouse antibody (sc-8439), anti-TNF-α rabbit antibody (sc-8301), anti-IL-6 rabbit antibody (sc-1265-R), anti-NF-κB p65 rabbit antibody (sc-372), anti-pNF-κB rabbit antibody (sc-101748), and anti-GAPDH mouse antibody (sc-365062) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary antibodies (anti-rabbit IRDye-800CW and anti-mouse IRDye-800CW) were purchased from LI-COR Biosciences (Lincoln, NE, USA).