Jiaojiao Tian,Yingying Chen,Tao Jiang,Xueyan Jia,Hui Gong,Xiangning Li
Resin embedding combined with ultra-thin sectioning has been widely used in microscopic and electron imaging to acquire precise structural information of biological tissues. However, the existing embedding method was detrimental to quenchable fluorescent signals of precise structures and pH-insensitive fluorescent dyes. Here, we developed a low-temperature chemical polymerization method named HM20-T to maintain weak signals of various precise structures and to decrease background fluorescence. The fluorescence preservation ratio of green fluorescent protein (GFP) tagged presynaptic elements and tdTomato labeled axons doubled. The HM20-T method was suitable for a variety of fluorescent dyes, such as DyLight 488 conjugated Lycopersicon esculentum lectin. Moreover, the brains also retained immunoreactivity after embedding. In summary, the HM20-T method was suitable for the characterization of multi-color labeled precise structures, which would contribute to the acquisition of complete morphology of various biological tissues and to the investigation of composition and circuit connection in the whole brain.
Summary Pancreatic stellate cells (PSCs) are suggested to play an important role in the development of pancreas and islet fibrosis. However, the precise contributions and solid in vivo evidence of PSCs to the fibrogenesis remain to be elucidated. Here, we developed a novel fate-tracing strategy for PSCs by vitamin A administration in Lrat-cre; Rosa26-tdTomato transgenic mouse. The results showed that stellate cells give rise to 65.7% of myofibroblasts in cerulein-induced pancreatic exocrine fibrosis. In addition, stellate cells in islets increase and contribute partly to myofibroblasts pool in streptozocin-induced acute or chronic islet injury and fibrosis. Furthermore, we substantiated the functional contribution of PSCs to fibrogenesis of pancreatic exocrine and islet in PSCs ablated mice. We also found stellate cells’ genetic ablation can improve pancreatic exocrine but not islet fibrosis. Together, our data indicates that stellate cells are vital/partial contributors to myofibroblasts in pancreatic exocrine/islet fibrosis.
Two trypanosomatid species, Lotmaria passim and Crithidia mellificae, have been shown to parasitize honey bees to date. L. passim appears to be more prevalent than C. mellificae and specifically infects the honey bee hindgut. Although the genomic DNA has been sequenced, the effects of infection on honey bee health and colony are poorly understood. To identify the genes that are important for infecting honey bees and to understand their functions, we applied the CRISPR/Cas9 system to establish a method to manipulate L. passim genes. By electroporation of plasmid DNA and subsequent selection by drug, we first established an L. passim clone expressing tdTomato or Cas9. We also successfully disrupted the endogenous miltefosine transporter and tyrosine aminotransferase genes by replacement with drug (hygromycin) resistant gene using the CRISPR/Cas9-induced homology-directed repair pathway. The L. passim clone expressing fluorescent marker, as well as the simple method for editing specific genes, could become useful approaches to understand the underlying mechanisms of honey bee-trypanosomatid parasite interactions.
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