Triphenyl tetrazolium chloride (ttc)

Coronary stenosis was created in 11 domestic, 3-month old pigs weighing 30–35 kg by implanting a bottleneck stent in the proximal LAD coronary artery as described previously [19 (link)]. In addition, a sham group of 4 pigs underwent a catheterization procedure without the stent implantation. Myocardial [⁶⁸Ga]NODAGA-RGD uptake was evaluated by PET 13 ± 4 days after the stent implantation. In the same imaging session, myocardial perfusion was quantified using [¹⁵O]water PET (physical half-life 2 min) at rest and during adenosine-induced stress before a [⁶⁸Ga]NODAGA-RGD (physical half-life 68 min) injection to localize the myocardium and to determine the ischemic area. Three pigs without a perfusion defect during adenosine stress were considered as procedural failures and excluded from further in vivo analyses.
Subsequently, the pigs were euthanized and their hearts were excised. Based on 1% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, Saint Louis, MO, USA) staining, tissue samples were obtained from the injured ischemic (TTC negative) and adjacent viable ischemic (TTC positive) myocardium as well as from the remote non-ischemic myocardium. Autoradiography of myocardial tissue sections was used to compare [⁶⁸Ga]NODAGA-RGD uptake with the histology, α_vβ₃ integrin expression and CD31 on endothelial cells.

The infarct volume was assessed at 6 h and 1–14 days after PTS. Mice were deeply anesthetized with sodium pentobarbital (50 mg/kg), and their brain tissues were collected and stored in a −20 °C refrigerator for 20 min before being sliced into sections that were 2 mm thick in the coronal plane. The brain sections were then stained with 2% TTC (Sigma-Aldrich) for 30 min at 37 °C in the dark, with the slices being turned evenly every 8 min to ensure even contact with the TTC staining solution. Subsequently, the tissues were fixed in a 4% paraformaldehyde (PFA) solution to ensure optimal tissue hardness. After staining, the infarct appeared white, while the normal brain tissue was stained red. We measured the infarct area using Image J (Version 2.0.0) as described [17 (link),18 (link)], and calculated the brain infarct volume percentage (BIVP).

Viability of brain ischemia was evaluated using TTC (Sigma-Aldrich, St. Louis, Missouri, USA) 24 h after MCAO. Rat brains were rapidly removed, frozen at −80 °C for 5 min and then sliced coronal into serial 2-mm-thick slices at the level of the bregma. Sets of five serial slices from each brain were incubated for 30 min at 37 °C in 2 % TTC (Sigma Co., St Louis, MO, USA) in the dark washed in PBS, and then fixed by 4 % formaldehyde in PBS. Images of sections from the exact center of the forebrain were captured using digital camera system (Leica, Solms, Germany). The infarction area and hemisphere area of each section were traced and measured using Image Pro plus 6.0 software (Media Cybernetics, Inc., MD, USA). To eliminate the interference of brain edema, the infarct volume was corrected by standard methods (contralateral hemisphere volume–volume of non-ischemic ipsilateral hemisphere). Infracted volume was expressed as a percentage of the contralateral hemisphere, as described previously [31 (link), 32 (link)].

This experimental performed from July 2019 to March 2020. The chemicals used in this study include culture medium de man, rogosa and sharpe (MRS), mueller hinton agar (MHA) and mueller hinton broth (MHB), compounds such as iron chloride, butylated hydroxy anisole (BHA), triphenyltetrazolium chloride, sodium acetate, tetrahydrofuran potassium hydroxide, trichloroacetic acid, and ammonium sulfate were purchased from Merck, Germany. Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), and penicillin and streptomycin antibiotics were developed from Gibco.

Agar, yeast nitrogen base w/o amino acids (YNBD), bacto-peptone and yeast extract were purchased from DifcoTM, Sweden. Additionally, glucose, ethanol p.a., triphenyl tetrazolium chloride (TTC) and dimethyl sulfoxide (DMSO) were purchased from Merck, Brazil. 4-Nitroquinoline-1-oxide (4-NQO) and canavanine (Can) were purchased from Sigma, New York, NY, USA. Phosphate-buffered saline (PBS) powder was purchased from Laborclin, Brazil. 4-NQO powder was dissolved in 10% ethanol solution and used as experimental positive control. PBS powder was dissolved in distilled H₂O, sterilized by autoclaving (20 min, 121 °C), stored at room temperature (PBS solution). This was used to wash cells and as experimental negative control. In this study, 1% DMSO solution (1% DMSO) was 1% in distilled H₂O.