Related relative mRNA expression was calculated by the 2 2?Cq method and normalized to -actin . was confirmed in MLE-12 cells Cell Death Detection Kit (Roche, USA) according to the manufacturers instructions. Briefly, sections (4?m) were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, rehydrated and heated for antigen retrieval. Endogenous peroxidase was blocked in 3% hydrogen peroxide. Three different dilutions (1: 7, 1: 11, and 1: 16) of terminal deoxynucleotidyl transferase (TdT) in reaction buffer (made up of a fixed concentration of digoxigenin\labelled nucleotides) were applied to serial sections at 37C for 1?hr before the slides were placed in Stop/Wash buffer for 10?min. Following intensive washing, a pre-diluted anti\digoxigenin peroxidase\conjugated antibody was applied for D-erythro-Sphingosine 30?min. Apoptotic cells were detected after incubation in the 3,3\diaminobenzidine (DAB) chromogen (DAKO, Carpinteria, CA, USA) for 6?min and slides were counterstained with Methyl Green (Sigma, St Louis, MO, USA). The presence of TUNEL+ cells was decided using the Image Analysis Software (Olympus, Japan). We evaluated the percentage of TUNEL+ cells relative to the total cells in the same area between the control and experimental groups D-erythro-Sphingosine (N?=?8 lungs for each group). Western blotting Western blotting was performed in accordance with a standard procedure. Protein samples were extracted from lung tissue homogenate or MLE-12 cell lysate using a radio-immuno-precipitation assay buffer (RIPA, Sigma, USA) supplemented with protease and phosphatase inhibitors, and the protein concentrations were quantified using BCA assay. The protein samples were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, MA, USA). The membrane was D-erythro-Sphingosine blocked with 5% non-fat milk and then was incubated with primary antibodies at 4C overnight against P65 (1:1000; Bioworld), SP-C (1:1000; Abcam), AQP5 (1:1000; Abcam), GATA-6 (1:1000; Abcam) or ABCA3 (1:1000; Abcam) in TBS buffer, respectively. -actin was used as a loading control (1:3000; Proteintech). After incubation with HRP conjugated secondary antibodies (1:3000; EarthOx), the blots were developed with the SuperSignalTM West Femto Chemiluminescent Substrate (ThermoFisher, Rockford, USA) and Gel Doc? XR+?System (BIO-RAD, CA, USA). The intensity of the bands was analyzed using the Quantity One software (BIO-RAD, CA, USA) according to the manufacturers instructions. The Western blotting results are representative of three impartial experiments. RNA isolation and qpcr Total RNA was isolated from fresh embryonic chick lung tissue or MLE-12 cells using the E.Z.N.A? Total RNA Kit (OMEGA, Georgia, USA) according to the manufacturers instructions. First-strand cDNA synthesis and the SYBR? Green qPCR assay were performed using the PrimeScriptTM RT Reagent Kit (Takara, Japan). All the specific primers used are described in Table 1. The reverse transcription reactions were performed in the Bio-Rad S1000TM thermocycler (Bio-Rad, USA). qPCR mixture was then incubated at 95C for a 3?min initial denaturation step UCHL2 followed by 40 PCR cycles (95C for 5s, 60C for 20?s, and 72C for 20?s), using the ABI 7000 Real Time PCR machines. Corresponding relative mRNA expression was calculated by the 2 2?Cq method and normalized to -actin . The D-erythro-Sphingosine qPCR results are representative of three impartial experiments. Table 1. Sequences of qPCR primers. model to further verify the connection between oxidative stress and LPS-induced abnormal pulmonary cell differentiation. LPS induced an intracellular ROS production enhancement, which was partially blunted by the addition of vitamin C to the culture medium (Physique 5C). GATA-6 plays a crucial role in regulating pulmonary epithelial differentiation during lung development, and LPS significantly inhibited GATA-6 expression in MLE-12 cells, while it was partially restored by vitamin C D-erythro-Sphingosine (Physique 5D, E). Western blotting and qPCR data further confirmed that LPS induced down-regulation of SP-C, ABCA3 and GATA-6 expressions, which could be restored by vitamin C (Physique 5F-H). These data exhibited that LPS could induce the oxidative stress, which subsequently led to the restriction to embryonic lung development. Open in a separate window Physique 5. Oxidative stress involved in LPS-induced abnormal development of chick lungs. (A, B): In immunofluorescent staining of E18 chick lungs, red color shows Nrf2 and blue color shows DAPI staining, respectively. The far right panel indicates the dotted squares of the merged images with higher magnification. LPS induced a dramatic increase in Nrf2 expression in E18 chick lungs. (C): Flow cytometry data show intracellular ROS production in MLE-12 cells was significantly increased by LPS. (D, E): In immunofluorescent staining of MLE-12 cells, red color.