Inactivation of horseradish peroxidase with crotonic acid for reprobing of western blotting.

Inactivation of horseradish peroxidase (HRP) treatment is a conventional preference to stripping for sequential detections of different proteins of chemiluminescent western blotting (WB). However, little evidence exists on whether other chemical substances treatment can affects the biological activity of HRP during stripping and re-probing of WB blots. Here, we successfully develop 20% crotonic acid (CA) as an alternative to stripping to inhibit HRP used for sequential chemiluminescent WB on polyvinylidene difluoride (PVDF) and Nitrocellulose (NC) membrane. Moreover, NC blots incubation in CA (40 °C, 30min) allow us to perform three round HRP inhibition in sequential detections without losing transferred proteins and damaging membrane. Hence, the method will help us save time and valuable samples without the need to rerun gels.

In vivo USPIO-enhanced MR signal characteristics of secondary degeneration in the ipsilateral substantia nigra after middle cerebral artery occlusion at 3T.

BACKGROUND AND PURPOSE: Ultrasmall superparamagnetic iron oxide (USPIO) particles to enhance MRI have been used to study neuroinflammation in vivo. Our purpose was to observe the USPIO-enhanced MR signal alterations in the primary ischemic lesion and ipsilateral substantia nigra after middle cerebral artery occlusion (MCAO) to verify the subsequent sequelae of neuroinflammation seen in the primary ischemic focus and secondary degeneration region. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to transient MCAO. In addition to conventional T2-, T1-weighted imaging, USPIO-enhanced MRI was performed in USPIO-injected stroke rats, while Gd-enhanced imaging was acquired in control stroke rats, on days 3, 6 using a 3-T MR scanner. The MR signal characteristics in the primary ischemic striatum, ipsilateral substantia nigra were noted, compared on histopathological H&E, Prussian blue (PB) staining. RESULTS: After MCAO, USPIO-induced T2 hypointensity changes were observed in the primary ischemic region with BBB impairment at both time points. In the substantia nigra ipsilateral to the primary ischemic lesion, there was no evidence of USPIO accumulation detected by MRI and PB staining, and no BBB leakage reflected by Gd-enhanced imaging on days 3 and 6. CONCLUSION: USPIO-enhanced MR signals have variable characteristics in both primary and remote sites after focal cerebral ischemia. This suggests that the neuroinflammatory response to brain ischemia in the primary ischemic focus and secondary degeneration region have different temporal patterns and pathophysiological mechanisms.