Eh0255

A total of 4 tissue sections (each 10-15 µm thick) were collected from each material and put into a 1.5 ml centrifuge tube. The samples were incubated in a 250 µl buffer (pH 7.5, 0.05 M Tris, 1 mM EDTA, and 0.5% Tween-20). Tissue samples were homogenized thoroughly and kept samples on ice for 30 min. Samples were Centrifuged at 10,000 x g for 20 min at 4˚C and the supernatant was used for measurement. Protein concentrations were measured with Bradford method (13 (link)). The levels of CX3CL1, CCL17, CXCR4, CXCL12 and ICAM5 were determined with the sandwich ELISA method in accordance with the manufacturer's protocols (cat. no. EH0255; Wuhan Fine Biotech Co., Ltd.) with inter-assay cv: <12% and intra-assay cv: <10%, respectively. IFN-γ and IRF5 values were presented as ng/µg protein. All ELISA measurements were performed using a microplate reader (BioTek Epoch). Results were presented as ml/mg of protein.

A total of four formalin-fixed, paraffin-embedded tissue sections (each 10- to 15-µm thick) were placed in a 1.5 ml centrifuge tube. Samples were incubated with 250 µl buffer (pH 7.5; 0.05 M Tris, 1 mM EDTA and 0.5% Tween 20). The tube was placed at 100°C for 10 min and immediately placed into a dry ice ethanol slurry until frozen. Protein extraction of all samples was performed as previously described (18 (link)). Protein concentrations were measured using the Bradford method (19 (link)). POSTN levels were measured with the sandwich ELISA method following the kit manufacturer's instructions (cat. no. EH0255; Wuhan Fine Biotech Co., Ltd.) with an inter-assay coefficients of variability (cv) of <12% and an intra-assay cv of <10%, respectively. The mean minimum detectable quantity of human POSTN was 0.094 ng/ml. POSTN values are presented as ng/µg protein. All ELISA measurements were performed using a microplate reader (BioTek Instruments, Inc.).