Search

Objective：To compare the effects of different fixation methods on primary cilia immunofluorescence in mammalian cells and to investigate the optimized strategy of primary cilia immunofluorescence imaging. Methods：Cells were fixed with 4% paraformaldehyde (PFA), methanol (MeOH) or 10% trichloroacetic acid (TCA), followed by the same conditions of penetration, blocking and antibody incubation, and finally the primary cilia imaging results were compared under the same shooting parameters. Results：PFA fixation resulted in the better imaging of ciliary axoneme proteins and part of the ciliary membrane proteins, and MeOH fixation showed more clear imaging of the proteins localized at the bottom of cilia. Compared with other two methods, TCA fixation was suitable for some ciliary membrane proteins and axoneme binding proteins although the staining signal of ciliary axoneme was weak. In addition to the variations of fluorescence intensity, there were also minor differences in protein localization observed by staining after fixation using different methods. Conclusion：Each of the three fixation methods has their own advantages and limitations in immunofluorescent staining of primary cilia, and the appropriate fixation method needs to be selected with comprehensive consideration of the experimental purpose and the characteristics of target protein. [ABSTRACT FROM AUTHOR]

In order to further visualize intracellular dynamics, precise imaging of endogenous proteins in live cells was performed using an antigen-binding fragment (Fab)-based Quenchbody (Q-body). The transfected Q-body probe showed an antigen-dependent fluorescence response, enabling the clear visualization and sorting of cells expressing p53, a tumor suppressor biomarker. [ABSTRACT FROM AUTHOR]

Porcine circovirus type 3 (PCV3), which currently lacks effective preventive measures, has caused tremendous economic losses to the pig husbandry. Obtaining the strain of PCV3 is the key to preparing related vaccines and developing corresponding antiviral drugs. In this study, according to the linear sequence of PCV3 DNA published on GenBank, the sequence was rearranged with SnapGene gene-editing software, and after rearrangement, the HindIII restriction endonuclease site was added to the end of the linear DNA, so that both ends have the same restriction endonuclease site. On this basis, the rearranged linear DNA is obtained by gene synthesis, PCR amplification, DNA purification, etc., and is digested and connected in vitro to obtain cyclized DNA. PCV3 infectious clones were obtained by transfecting 3D4/21 cell lines. The obtained PCV3 was identified by PCR, Western blotting, and indirect immunofluorescence tests. The results showed that this study successfully obtained the strain of PCV3 in vitro. To further evaluate the pathogenicity of the obtained PCV3 infectious clones, this study established an animal model of Kunming mice infected with PCV3. The results of RT-PCR, Western blotting and immunohistochemistry showed that PCV3 can infect myocardium and alveoli of Kunming mice, but no PCV3 was detected in other tissues. The above studies indicate that PCV3 circular DNA can be used to construct PCV3 infectious clones. This research will provide a new method for the construction of circular DNA viruses and lay the foundation for the research and pathogenesis of PCV3 vaccine.

Porcine circovirus type 3 (PCV3), which currently lacks effective preventive measures, has caused tremendous economic losses to the pig husbandry. Obtaining the strain of PCV3 is the key to preparing related vaccines and developing corresponding antiviral drugs. In this study, according to the linear sequence of PCV3 DNA published on GenBank, the sequence was rearranged with SnapGene gene-editing software, and after rearrangement, the Hin dIII restriction endonuclease site was added to the end of the linear DNA, so that both ends have the same restriction endonuclease site. On this basis, the rearranged linear DNA is obtained by gene synthesis, PCR amplification, DNA purification, etc., and is digested and connected in vitro to obtain cyclized DNA. PCV3 infectious clones were obtained by transfecting 3D4/21 cell lines. The obtained PCV3 was identified by PCR, Western blotting, and indirect immunofluorescence tests. The results showed that this study successfully obtained the strain of PCV3 in vitro. To further evaluate the pathogenicity of the obtained PCV3 infectious clones, this study established an animal model of Kunming mice infected with PCV3. The results of RT-PCR, Western blotting and immunohistochemistry showed that PCV3 can infect myocardium and alveoli of Kunming mice, but no PCV3 was detected in other tissues. The above studies indicate that PCV3 circular DNA can be used to construct PCV3 infectious clones. This research will provide a new method for the construction of circular DNA viruses and lay the foundation for the research and pathogenesis of PCV3 vaccine. [ABSTRACT FROM AUTHOR]