Image labtm analysis software

PrP^Sc extraction and the following WB analyses were carried out on the brainstem samples collected at necropsy from the experimentally challenged goats. Brain tissue from healthy cattle, C-BSE and L-BSE samples, used for the inoculum, and goats previously confirmed positive or negative for classical scrapie were also analyzed as controls. The analyses were performed as previously reported^{11 (link)}. PrP^Sc was detected using SAF84 (mouse; Cayman Chemical Co.; Cat. No. 189775) monoclonal antibody and an anti-mouse antiserum conjugated with alkaline phosphatase. Reaction was revealed by a chemiluminescent substrate and visualized on Hyperfilm ECL (Amersham GE-Healthcare, Life Sciences) or by a gel documentation (UVI Prochemi, Uvitec). Image Lab^TM analysis software (Bio-Rad) was used to analyse blot images.

Activities of MMP-2 and MMP-9 were determined in the N9 extracellular media, either alone or after incubation with LPS, GUDCA or VS, by performing a SDS-PAGE zymography in 0.1% gelatin-10% acrylamide gels, under non-reducing conditions (Silva et al., 2010 (link)). Briefly, after electrophoresis, the gels were washed for 1 h in a solution containing 2.5% Triton-X-100 to remove SDS and to renature the MMP species in the gel and then incubated at 37°C to induce gelatin lysis (buffer: 50 mM Tris pH 7.4, 5 mM CaCl₂, 1 μM ZnCl₂) overnight. Gels were then stained with 0.5% Coomassie Brilliant Blue R-250 (Sigma-Aldrich) and destained in 30% ethanol/10% acetic acid/H₂O (v/v). Gelatinase activity, detected as a white band on a blue background, was measured using the Image Lab^TM analysis software (Bio-Rad).

Cells were collected in Cell Lysis Buffer, as usual in our lab (Vaz et al., 2015 (link)). Briefly, total cell extracts were lysed for 5 min on ice with shaking, collected with cell scrapper, and sonicated for 20 s. The lysate was then centrifuged at 14,000 g for 10 min at 4°C, and the supernatants were collected and stored at −80°C. Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA) according to manufacturer's specifications. Then, equal amounts of protein were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% (w/v) nonfat milk solution, membranes were incubated with primary antibody mouse anti-HMGB1 (1:200, from Biolegend) diluted in 5% (w/v) BSA overnight at 4°C, followed by the secondary antibody goat anti-mouse HRP-linked (1:5,000, sc-2005, Santa Cruz Biotechnology®) diluted in blocking solution. Chemiluminescence detection was performed by using LumiGLO® reagent (Cell Signaling) and bands were visualized in the ChemiDoc^TM XRS System (Bio-Rad). The relative intensities of protein bands were analyzed using the Image Lab^TM analysis software (Bio-Rad).