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제목 Itions were 95 for 10 min to activate DNA polymerase, followed by 45 …
작성자 Fausto Baskin / 23-09-28 13:57
작성일 23-09-28 13:57

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Itions were 95 for 10 min to activate DNA polymerase, followed by 45 cycles of 95 for 15 s, 60 for 15 s, and 72 for 15 s. Specificity of amplification products was determined by melting curve analysis. The qRT-PCR reactions for each sample were repeated three times. Independent experiments were done in triplicate.Immunohistochemistry and evaluation of stainingTotal RNA was extracted from the cell lines and lung tissues using Trizol (Takara, Shiga, Japan). RNA (1 g) was reverse transcribed into cDNA, and cDNA was used as a template to amplify with specific primers for sense: 5-TCAATGGCGGTTCTCATGCT-3 and for antisense: 5-GCAGCTCCAGGCCTTCTTTA-3. ARF5 was used as an internal control with primers for sense: 5ATCTGTTTCACAGTCTGGGACG-3 and for antisense: 5-CCTGCTTGTTGGCAAATACC-3. Experiments D(+)-Galactosamine (hydrochloride) wereImmunohistochemistry was performed in primary NSCLC tissues, non-cancerous lung tissues, and mouse tumors according to a previous description [44] with rabbit polyclonal anti-ENO1 antibody (1:150; Proteintech, USA), anti-cyclin D1, p21 antibody (1:100; Epitomics, Burlingame, USA), -catenin, N-cadherin, or E-cadherin antibody (1:100; Cell Signaling Technology, Danvers, USA). Stained tissue sections were reviewed and scored independently by two investigators blinded to the clinical data. For cytoplasmic staining, the score was based on the sum of cytoplasm staining intensity and the percentage of stained cells. The staining intensity was scored as previously described (0?) [44,45], and the percentage of positive staining areas of cells was defined as a scale of 0? (0: 76 ). For nuclear staining, the staining score was defined based on the sum of nuclear staining intensity and the percentageFu et al. Journal of Hematology PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6833145 Oncology (2015)8:Page 11 ofof positive nuclear staining. Positive nuclear staining scores were defined as follows: 0: 80 . The sum of the staining intensity and staining extent scores (0?) was used as the final staining score. For statistical analysis, a final staining score of 0 2 and 3 6 in cytoplasm or 0 3 and 4 6 in nucleus were respectively considered to be negative and positive expression levels. Expression of ENO1 in the nucleus was observed, but since ENO1 localizes in cytoplasm, only the cytoplasmic staining was evaluated.ImmunofluorescenceTable 2 shRNA sequences for ENOshENO1 A Sense Antisense B Sense Antisense C Sense Antisense Sequence 5-CCGGAATGTCATCAAGGAGAAATATCTCGAG ATATTTCTCCTTGATGACATTTTTTTG-3 5-AATTCAAAAAAATGTCATCAAGGAGAAATAT CTCGAGATATTTCTCCTTGATGACATT-3 5-CCGGCGTGAACGAGAAGTCCTGCAACTCGA GTTGCAGGACTTCTCGTTCACGTTTTG-3 5-AATTCAAAACGTGAACGAGAAGTCCTG CAACTCGAGTTGCAGGACTTCTCGTTCACG-3 5-CCGGCCACTGTTGAGGTTGATCTCTCTCGAGA GAGATCAACCTCAACAGTGGTTTTTG-3 5-AATTCAAAAACCACTGTTGAGGTTGATCTCT CTCGAGAGAGATCAACCTCAACAGTGG-Immunofluorescence was performed according to a previous study [46]. NSCLC cells were seeded on coverslips in six-well plate and cultured overnight. Subsequently, cells were fixed in 3.5 paraformaldehyde and permeabilized in KB solution and 0.2 Triton X-100 at room temperature. After the blocking solution was washed out, cells were incubated with a primary antibody (ENO1) (diluted in KB) for 30?5 min at 37 and subsequently washed with KB twice. After incubating for 30?5 min at 37 with secondary antibody (diluted in KB) and washing with KB again, the coverslips were then mounted onto slides with mounting solution containing 0.2 mg/ml D.