2 3 5 triphenyl tetrazolium chlorides

Hydrogenated soy phosphatidylcholine (HSPC) was ordered from lipoid (USA). Stearylamine (SA) and cholesterol (Chol) and 2,3,5-triphenyl tetrazolium chlorides (TTC) were purchased from Sigma (St Louis, MO). Vancomycin was obtained from Dana Pharmaceutical Company (Tehran, Iran). C2DA purchased from Santa Cruz (Texas, USA), chloroform, methanol, crystal violet, and glucose monohydrate were provided by Merck (Darmstadt, Germany). Trypticase soy agar (TSA), trypticase soy broth (TSB), and Mueller Hinton broth (MHB) were purchased from Himedia (Mumbai, India).

The cryostat was used to cut the frozen brain tissue into brain sections with a thickness of about 2 mm, which were placed in a tinfoil paper‐coated culture dish for cassette operation. Subsequently, an appropriate amount of 2% 2,3,5‐triphenyltetrazolium chlorides (Sigma Co.) was added to just immerse the sections, which was placed in a 37°C incubator for incubation for 30 min after covering the cassette lid. Then, the sections were washed with phosphate buffer saline (PBS) and fixed with 4% paraformaldehyde. The normal brain tissues were stained with bright red; if there is the infarction area, the area will not be stained and is pale due to decreased dehydrogenase activity. The images were imported into ImageJ software (version 1.52a, NIH) to quantify the cerebral infarction ratio and brain swelling.

The rats were anesthetized with isoflurane and decapitated at 24 h post HI. The brains were rapidly removed and sliced into 2-mm-thick coronal sections in a rat brain matrix (Seino Co., Ltd., Beijing, China). The sections were immediately immersed in 2% 2,3,5-triphenyltetrazolium chlorides (Sigma Co., St. Louis, MO, United States) at 37°C for 30 min in the dark, washed in PBS and then fixed by 4% formaldehyde in phosphate buffered solution. The infarction area of each section was traced and measured using Image J Software (Version 1.43 u; National Institutes of Health, Bethesda, MD, United States). To abolish the error caused by brain edema, we corrected the infarct volume by standard methods as described in the previous report (contralateral hemisphere volume – volume of non-ischemic ipsilateral hemisphere), with infracted volume expressed as a percentage of the contralateral hemisphere. In addition, the brain swelling was determined by subtracting the total volume of the non-ischemic hemisphere from that of the ischemic hemisphere (Hu et al., 2009 (link)).