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.