Quantitative Real-time PCR
For checking the expression level of targeted mRNA in HEK293T, total RNA was extracted from transfected HEK293T cells using Trizol (Tiangen, China) according to the protocol of the manufacturer. 1 μg of RNA was used for cDNA synthesis by using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time, TAKARA, Japan) or TIANScript ⅡRT Kits (KR107, TIANGEN, China). Quantitative real-time PCR was performed using the TB Green® Premix Ex Taq™ II (Tli RNaseH Plus, TAKARA, Japan). The relative expression levels of genes were calculated and quantified by the method of ΔΔCt.
Immunohistochemical analysis
Immunohistochemical (IHC) staining was performed as follows: paraffin sections were successively placed in xylene (two times), 100% ethanol (two times), 95% ethanol, 85% ethanol, 75% ethanol, 70% ethanol, and PBS (two times), for 5 minutes each for dewaxing. The sections were incubated in 3% hydrogen peroxide for 10 minutes, and washed twice with PBS. Then, the sections were placed in antigen repair solution preheated to 100 ℃ and heated for another 20 minutes. Sections were blocked with block solution for 25 minutes and incubated with the primary antibody (polyclonal anti-mcherry, 26765-1-AP, proteintech, China) overnight. After washing with PBST and PBS, sections were incubated with secondary antibody (HRP-labeled Goat Anti-Rabbit IgG(H+L), A0208, Beyotime, China) for 1 hour, and washed with PBST and PBS. DAB chromo developer (Beyotime, China) was prepared and added to the sections by drops. After chromo development for about 3 minutes, the sections were rinsed with PBS. Hematoxylin was applied for 1 minute, and then the sections were rinsed with water. The slices were successively dehydrated in 70%, 75%, 80%, 95%, 100% ethanol (two times), and xylene (two times) for 5 minutes each, and then placed in a ventilated place to dry. Finally, the slices were sealed with neutral resins (Solarbio, China) and preserved.