A method by using ?-BT-HRP conjugate to localize N-acetylcholinc receptor of neuromuscular junction was described.Thin strips of fresh muscle were incubated with ?-BT-HRP conjugate at a concentration of 1?10-7 M in Tyroid's solution and then reacted with Karnovsky's DAB medium.There were obviously brownish red positive deposits to constitute various vesicular forms seen under a light microscope. Under an electron microscope we can see positive deposits that were localized both on the presynaptic and postsynaptic membra-nes. The binding activity and reliability of a-BT-HRP were discussed.

We report in detail ultrastructural changes and freezing damage mechanism about heart muscle and some organelles after rapid freezing. The ventricles of rat heart were cut pieces about 100-150/?m by microsiicer. The pieces were quickly injected liquid cryogen Freon 22 by Reichert-Jung spring-assistant mechanism (KF-80). The specimens frozen were rapidly transferred into substitution medium aceton and kept at -80℃(28h), then -60℃ (48h),-20℃(12h)and 4℃ (1 h). The structures of specimens frozen were well and there were no ice crystals in the area of the tissue frozen surface to 20?m depth. However, there were freezing damages in mitochondrial crista, intercellular substance and muscular fibre in the tissue surface to 30?m depth. The structure of tissue was destroyed by ice crystal over 50?m depth in the tissue. The results suggest that intercellular substance and mitochondrial crista are the most sensitive to ice crystal damage after rapid freezing of heart tissue, then the less sensitive are muscular fibre and nucleus. The unit membrane is not easy to be damaged by ice crystal.

Objective To investigate the effect of complement C3a on mouse podocytes phenotype transformation.Methods Purified C3a recombinant protein was used to stimulate mature mouse podocytes.The expression of the mature podocyte markers synaptopodin,podocin,nephrin,CD2-associated protein (CD2AP) and the mesenchymal cell markers fibroblast specific protein 1 (FSP-1),α-smooth muscle actin (α-SMA) were detected by RT-PCR,Western blotting,immunochemistry and immunofluorescence,respectively.Some podocytes were transfected with integrin-linked kinase (ILK) siRNA before the administration of C3a,the expression of nephrin and α-SMA were accessed by Western blotting,and the expression of Snail and α-actinin 4 were accessed by Western blotting and immunochemical method.The migration ability of podocytes was observed by scratch test.Results Immunocytochemistry and immunofluorescence analysis showed that synaptopodin,podocin,nephrin,CD2AP were highly expressed by mature mouse podocytes.The expression of these podocyte markers could be markedly inhibited after 24 h of C3a (0.1 μmol/L) treatment,and accompanied by the induction of mesenchymal markers FSP-1 and α-SMA.Compared with control group,the mRNA levels of synaptopodin,podocin,CD2AP and nephrin were significantly repressed by the administration of C3a in a dose-dependent manner,whereas the transcription of FSP-1 and α-SMA were remarkably up-regulated by C3a treatment (P ＜ 0.05,respectively).Western blotting analysis also confirmed the decrease of synaptopodin,podocin,nephrin and CD2AP protein and the increase of FSP-1 and α-SMA protein were closely depend on the C3a concentration (P ＜ 0.05,respectively).To further assess the downstream of C3a,some podocytes were transfected with ILK siRNA before the administration of C3a.Compared with C3a group,the protein levels of nephrin and α-SMA were significantly changed by the administration of ILK siRNA (P ＜ 0.05,respectively).The expression of α-actinin 4 and Snail induced by C3a were inhibited by ILK knockdown (P ＜ 0.05,respectively),accompanied by a decline of cell migration potency.Conclusion Complement fragment C3a can induce transformation of mouse podocytes to mesenehymal cells,and ILK signaling pathway is involved in this cell type transformation.