Rnastore reagent

The experiment was divided into three groups, with fifty shrimp per group. To detect the distribution of LvCLEC4F, the hepatopancreas, gill, muscle, and eyestalk of healthy L. vannamei were collected. Three shrimp from every group were taken. The remaining L. vannamei were individually orally infected with the WSSV bait. The viral load of the WSSV bait was 1 × 10⁷ copies. The hepatopancreas, gill, muscle, and eyestalk of L. vannamei at 24, 48, 72, 96, 144, 192, and 228 h post-WSSV infection were collected and stored in RNAstore reagent (Tiangen Biotech Co., Ltd., Beijing, China) at −80 °C. Three individual shrimp from each group were taken at every sampling time. The collected samples were used for total RNA extraction and cDNA synthesis to assess the expression level of LvCLEC4F.

Rats were euthanized with an overdose of isoflurane (5% isoflurane with 2 L/min oxygen). The viscera (lung, heart, liver, and spleen) were rapidly dissected, and then the heart was separated into the RV and the left ventricle (LV) with interventricular septum (IVS) and then weighed separately. Finally, the viscera weight/body weight and the ratio of the weight of right ventricular wall to the weight of left ventricular wall plus IVS [RV/(LV+IVS)] were measured and calculated as indices of right ventricular hypertrophy.
After weighing, the upper left lung and heart were fixed in 4% buffered paraformaldehyde and underwent hematoxylin/eosin staining. Pulmonary arterial vascular remodeling was blindly measured by the % of wall thickness (total diameter–internal diameter/total diameter) through H&E staining at 200× magnification, with five arteries per lung section. The heart was stained using TUNEL (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Apoptotic cells were counted under a microscope at 400× magnification, and five regions were randomly selected from each section. The apoptosis rate was expressed as the ratio of apoptotic cells to total cells in the same visual field. The RV was divided and stored at -80°C for immunoblotting or placed t in RNAstore reagent (TIANGEN Biotech, Co. Ltd., Beijing, China) for PCR analysis.

Rat kidneys were collected immediately after being sacrificed. The cortex of the right kidneys from three rats in each group was dissected and stored in RNA store reagent (Tiangen Biotech, Beijing, China) at 4°C overnight and later stored in -80°C for RNA extraction and transcriptome sequencing. The remaining kidney tissues were fixed in 4% paraformaldehyde and paraffin-embedded. The tissue sections (4 μm) were stained with hematoxylin and eosin (HE), Masson trichrome, and Periodic Acid-Schiff (PAS) and examined under a light microscope. At least 5 random positive areas in each section were photoimaged (magnification ×400) and analyzed using ImageJ (National Institutes of Health, USA).

CRC tissues and matched adjacent tissues were surgically resected from six patients with CRC and were obtained from the Second Hospital of Hebei Medical University, Shijiazhuang, China. The diagnosis and histological Evaluation of CRC were performed by two pathologists. After excision, tissues were immediately placed at −80 °C, or immediately stored in RNAstore Reagent (Tiangen, Beijing, China), or fixed in 4% paraformaldehyde (Shijiazhuang Huawo Kerui Biological Technology Co., Ltd., Hebei Province, China). This study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University (approval letter No. 2017- R028), and informed consent forms were obtained from all patients.

Heart tissues were stored in RNAstore Reagent (#Q5324, TIANGEN). Total RNA was extracted from heart tissues or cardiac myocytes using TRIzol reagent (Invitrogen, United States) according to the manufacturer’s instructions. cDNA was synthesized with the PrimeScriptTM RT Reagent Kit (TAKARA, Japan). RT-PCR was carried out using the SYBR Green premix Ex TaqⅡKit (Roche, Germany). Expression of PPARγ2, BNP, and β-MHC mRNA in heart tissues and BNP and ANP mRNA in cardiomyocytes was measured using RT-PCR. After the reaction system was completed, all gene levels were analyzed according to the Cq values. Primer sequences were as described in Table 1. The protocol of PCR was as follows: 95.0°C for 0.5 min, 95.0°C for 5 s, 60°C for 34 s, more than 39 cycles of 95.0°C for 5 s, with additional melt curve program included (Thornton and Basu, 2015 (link)).