Gel chemi doc system

The spinal cord in the lumbar enlargement was rapidly removed, and the ipsilateral dorsal spinal cord was separated and collected for Western blot analysis. Protein extraction was performed as described previously.^{15 (link)} Proteins (30 µg per sample) were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were blocked for 2 h in 1% bovine serum albumin (in 0.05% Tween-20 in Tris-buffered saline [TBST]) at room temperature and then immunoblotted overnight at 4°C with primary antibodies. After three washes in TBST, the membranes were incubated with goat anti-mouse IgG-horseradish peroxidase or goat anti-rabbit IgG-horseradish peroxidase secondary antibodies (Santa Cruz) for 2 h at room temperature. The membranes were then washed three times in TBST. Immunoreactive protein bands were detected using the Gel/Chemi Doc System (Bio-Rad).

To assess the relatedness of the strains, PFGE was performed as per the standard operating procedure for PulseNet PFGE for Escherichia coli O157:H7, CDC protocol (PLN05) (STEC 2021 ) with a CHEF-DR-III (Bio-Rad). The PFGE conditions were optimized, and the run time was set to 17.6 hours. The PFGE conditions involved an initial switch time of 2.16 s and a final switch time of 63.8 s. The Salmonella Braenderup strain H9812 (kindly gifted by the CDC, Atlanta, USA) was used as a molecular weight marker. Gel images were taken using the Gel/ChemiDoc system (Bio-Rad Laboratories). Analyses and comparisons of the PFGE fingerprints were performed using BioNumerics Software (5.1 version, Applied Maths).