Ttc solution

At 48 h after HI, the pups were deeply anesthetized with isoflurane. The brain was immediately isolated after perfusion with 10 ml ice-cold phosphate buffered saline (PBS) and stored at −20°C for 20 min and subsequently was sliced into 1.5 mm coronal sections. The sections were immersed in 2% TTC solution (Sigma-Aldrich, USA) at 37°C for 20 min in the dark. After washing with distilled H₂O, the sections were fixed in 4% paraformaldehyde for 20 min and photographed. The infarcted portions were identified as the white areas without TTC staining. The infarcted areas of each section were traced and measured using Image-Pro Plus 6.0 (U.S. National Institutes of Health, Washington, DC) (24 (link)). The total infarct volume was calculated by summation of the representative infarct volume of each section. The infarction percentage was calculated as follows: ([total contralateral hemispheric volume] – [total ipsilateral hemispheric stained volume]) / (total contralateral hemispheric volume × 2) × 100% (19 (link), 25 (link)).

Rats from a satellite cohort were sacrificed 1 day and 7 days after SKP-SCs transplantation, and cerebral infarction volume was measured after 2,3,5-triphenyltetrazolium chloride (TTC) staining. Briefly, after deep anesthesia, rats were transcardially perfused with saline. The brains were quickly removed, and the coronal slices cut at 2 mm with a rat brain mold (Shenzhen Ruiward Life Technology Co., China). These slices were immediately stained with 2% TTC solution (Sigma, USA) in PBS for 30 min in a 37°C incubator. Normal brain tissue is stained red, while infarcted areas remain unstained. The size of the infarcted area was measured by Image J software (NIH, USA) and the infarct volume is expressed as the percentage of the infarcted brain volume in the lesioned (right, ipsilateral) hemisphere over the intact left, contralateral hemisphere (total brain volume of the intact left (contralateral) hemisphere minus the leftover healthy brain volume of the right (ipsilateral) hemisphere, divided by the total volume of the left hemisphere).

The cerebral infarct volume was assessed using TTC staining at 72 h post-BMSC transplantation. Brains (n = 4 per experimental group) were cut into 2 mm coronal slices and incubated in a 1% TTC solution (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min. White- and red-stained areas indicated ischemic and nonischemic tissues, respectively. The relative cerebral infarct volume in the brain tissue was analyzed using ImageJ software (NIH).

After 72 h reperfusion, mice were anesthetized with isoflurane (4–5%) followed by euthanasia using the cervical dislocation method. The brains were isolated and placed in a brain matrice (Kent Scientific Corp.), and seven 1 mm coronal sections were made from the olfactory bulb to the cerebellum using a pre-chilled razor blade. These slices were incubated in 1.5% 2,3,5-Triphenyltetrazolium chloride (TTC) solution (Sigma, St. Louise, MO, USA) at 37 °C in an incubator for 10 min. The stained brain section images were captured with a digital scanner and transported to a computer. The infarct area of each brain slice was measured in a blinded manner using ImageJ software (National Institutes of Health). The infarct volume was calculated by Swanson’s method to correct for edema [17 (link)]. The total volumes of both contralateral and ipsilateral hemisphere, and the volumes of the striatum and cortex in both hemispheres, were measured, and the infarct percentage was calculated as % contralateral structure to avoid mismeasurement secondary to edema.

Triphenyltetrazolium chloride (TTC) staining was performed 3 days after MI. Each mouse was kept under deep anesthesia using isoflurane (5%), the heart excised, and cut into five transverse slices with equivalent thickness. These slices were incubated in 1% TTC solution (Sigma Aldrich, St. Louis, MO) at 37°C for 10 min and photographed under the microscope (Leica, M165; Wetzlar, Germany). On each image, we measured the infarct area (i.e., the area lacking TTC staining) and ventricular wall areas using Image-Pro software (version 10; Media Cybernetics, Rockville, Maryland). The ratio of the net infarct area in the net ventricular wall area was calculated from five slices.