The Fine Structure Of Zebrafish Spleen,Heart And Body Kidney And The Standardization Of Sample Preparation

Zebrafish(Danio rerio)is a small,tropical teleost with a small size,easy reproduction,short reproductive cycle,clear genetic background,in vitro fertilization,and embryonic developmental transparency,zebrafish and human genes have a high degree of homology of 87%as well.Due to its high degree of homology,zebrafish has become a model animal after mice,drosophila,and nematodes,which has been widely used in genetic development,tumor biology,molecular biology,drug screening,toxicity testing,and environmental monitoring.and many other areas.Spleen is an important immune organ in the zebrafish,it also has functions of hematopoiesis,blood storage and immunity.Heart is the most important organ for energy supply in the zebrafish,it is closely related to the substance transport,internal stability and defense function in the fish.Body kidney is the main excretory organ in the zebrafish and participates in the excretion of various metabolic substances in the fish.So far,there have been few studies on zebrafish spleen,and most of them focus on the toxicity test.There are relatively many studies on the heart and body kidney of zebrafish,but most of them explore heart development,endocrine conditions,and kidney disease models establishment from the genetic level.This study will describe and interpret the zebrafish spleen,heart and body kidney tissue structure from the morphological point of view,and provide normal morphological reference for relevant pathological toxicology studies.Experiment ? The fine structure of zebrafish spleenThe fine structure of zebrafish spleen were observed and analysis by light and transmission electron microscopy.The results showed that the spleen parenchyma of the zebrafish was composed of splenic pulp and reticular tissue,the splenic pulp was distributed throughout the spleen,wbich including white pulp and red pulp,however,but the red and white marrow had no obvious boundaries,which present mixed distribution.The red pulp stain is shallow and has a large area,it accounts for most of the splenic parenchyma and is mainly composed of dense mature and immature red blood cells,nucleus is dark blue and the cytoplasm is light red.Splenic sinusoid structure is visible,which contains a large number of blood cells,the morphology and size of blood cell is not same,there is no obvious wall structure.Splenic cord structure is not obvious.The white pulp was darkly stained and consisted mainly of dense lymphocytes,the nucleus was darkly stained and dark purple.The outer surface of the spleen is covered with a thin layer of capsule,which is fibrillar,non-muscular,and no dense trabeculae,the capsule is mainly composed of medial monolayer squamous epithelium.Under electron microscope,the red pulp area of zebrafish spleen is relatively large,mainly composed of dense red blood cells.In addition to red blood cells,there are scattered platelets and plasma cells in the red pulp.The white pulp is mainly composed of lymphocytes.In addition to lymphocytes,there are many immune-related cells such as macrophages and monocytes in the white pulpExperiment ? The fine structure of zebrafish heartWe used light microscopy and transmission electron microscopy to observe and study the ultrastructure of zebrafish heart.The results showed that the zebrafish heart was composed of four parts:ventricle,atrium,bulbus arteriosus and venous sinus.The appearance of the ventricle was conical,thick-walled,muscular,covered by pericardium.Ventrical mainly composed of developed myocardial fibers,myocardial fibers are arranged in different directions and connected with each other to form the trabeculae,the trabeculae connected with each other to form a multi-lumen reticular structure to form a stent for the heart.Atrium is located at the upper right of the ventricle,the wall is thin and is filled with blood from all over the body,the atrial wall is mainly composed of thin,sparsely arranged muscle trabeculae(comb muscles),most of which originate in the atrioventricular valve and face the lateral wall.The appearance of the bulbus arteriosus is pear-shaped and consists of three layers:the outer layer,the middle layer,and the intima layer.Under electron microscopy,the ventricular stratification was obvious,which can be devided into dense layers of the epicardial region and sponge layer or porous layer of the endocardial region,dense layers is composed of longitudinally arranged flat myofibrils,sponge layer is composed of flat myofibrils arranged in different directions.There is no obvious stratification of the atrium,only the loose sponge layer,the surface covered with a thin layer of the capsule,the outer surface of the capsule has scattered caveolae distribution.The number of special particles in the atrium is much morer than ventricle,the number of interstitial cells in the atrium are also more than ventricle,and there are many cell types,compared with the ventricle.Under electron microscopy,bulbus arteriosus can be divided into three layers:intimal layer,middle layer and outer layer from the inside to the outside.The intimal layer is usually divided into finger-shaped stereoscopic ridges,which mainly composed of endothelial cells and smooth muscle cells.The middle layer is mainly composed of elastic fibers and smooth muscle cells,and the outer layer is mainly composed of thin mesothelial cells and collagen,collagen is mainly distributed in the area adjacent to the outer layer of the bulbus arteriosus.Experiment ? The morphological structure of zebrafish body kidney and the distribution and identification of its exosomesThe microstructure of zebrafish body kidney and the distribution and identification of exosomes in body kidney was studied by immunohistochemistry and immunofluorescence and transmission electron microscopy.The results showed that the kidneys consisted mainly of nephron and collecting ducts.The nephrons included the renal bodies.And the renal tubules,the renal body consists-of glomeruli and renal capsule,the renal tubules are divided into the neck,the proximal tubules and distal tubules.The free surface of the tubular lumen has a clear brush border by alkaline phosphatase staining.Under the electron microscope,the brush edge is composed of microvilli,and the appearance of the microvilli is elongated and tubular.Exosomes were strongly positive in the free surface of the tubular lumen,and glomerular and interstitial positivity were weak.Under the electron microscope,the distribution of late endosomes was observed on the free surface of the renal tubules,the distribution of multiple vesicles bodies was observed at the apical end of the renal tubules,multiple vesicles bodies were also observed in the glomerular podocytes,and multiple vesicles bodies in different parts of the kidney were observed with different electron density,the electron density of multiple vesicles bodies near the free surface is lower,and the electron density of t multiple vesicles bodies near the base is higher.Experiment ?The preparation procedures of light and transmission electron microscopy samples for zebrafish spleen,heart and body kidneyThe zebrafish is small in size,and difficult to obtain and prepare samples for organs such as spleen,heart,and body kidneys.In this section,we describe in detail the extraction and treatment methods of zebrafish spleen,heart and body kidney in the experiment,aiming to program the light and electron microscopy sample processing of zebrafish related organs.Research topics include TEM sample processing,staining,light microscopy sample processing,HE staining,Gordon Sweets reticular fiber staining,and acid phosphatase staining.Through the above process,the ideal observation results are obtained.It is helpful to standardize the operation of morphological observation sample processing of zebrafish organs and provide standardization operating steps for the extensive application of zebrafish in environmental monitoring and other fields.