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After blocking, plates were incubated with the mice serum samples in a serial dilution at 37°C for 1 h.
After blocking with PBST containing 1% casein, the membranes were incubated with mice serum samples (1/100 dilution).
All the mice serum samples were collected at the Department of Otolaryngology-Head and NeCaseurgery, Case Western Reserve University, University Hospitals-Case Medical Center.
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Two antibodies showed specific bands in the low molecular weight area of mouse serum samples.
Similarly, SEP54 antibody detected stronger signal in ob/ob mouse serum samples than that in WT mouse sample.
Mouse serum samples were obtained at 24 h after CLP injury, and the serum was separated by centrifugation at 3000 rpm for 15 min at 4 °C and then stored at −20 °C until use.
Analysis of the mouse serum samples revealed the presence of extremely low-level NAPQI-albumin adducts of approximately 0.2% of the total mouse serum albumin (MSA), regardless of the length of drug exposure.
After washing with PBS, diluted mouse serum samples collected after each immunization were incubated at 37 °C for 1 h, followed by washing three times, 100 μL of horseradish peroxidase (HRP -conjugated anti-mouse antibody at different dilutions (1:100, 1:1000, and 1:10,000) were added to wells.
Individual mouse serum samples were added in serial dilutions and incubated for 2 hours.
Mouse serum samples (100 µl, 1∶4 dilutions) were used for analyses with pre-immune serum as negative controls.
To determine systemic cathepsin levels, mouse serum samples from Cstc+/+ and Cstc−/− mice were used directly for ELISA.
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