An Improved Paraffin-embedded Method To Preserve Fluorescent Proteins For Large Tissues

Obtaining three-dimensional(3D)images of large samples is a challenge for life science.The three-dimensional imaging technologies are effective methods to obtain three-dimensional images of large volume samples.However,one of the challenges faced by these technologyies is how to embed large samples while achieving the purpose of maintain biological characterization information such as the morphological structure of complete tissues.Paraffin-embedded tissues have good sectioning properties and preservation of morphology which can produce thin sections to identify morphologic changes of tissues and cells.It seems that paraffin-embeded method has enormous potential to solve the question mentioned However,despite the continuing long-term success of paraffin-embedded,there are still some restrictions.One is the traditional formalin fixed paraffin embedding method(FFPE)only suitble for small tissues,limiting its ability to treat large specimens such as whole organs.The other one is quenching of fluorescence which makes it difficult to combine with the modern labelling techniques.In this study,we developed a paraffin-embedding method named by studying the influence of different parameters on embedding quality and fluorescence.We obtained continuous images of cell architecture and neural projections of the macaque brain with single-cell resolution.The main research contents and conclusions are as follows:(1)We develop the paraffin embedding method for large volume biological samples,and the maximum volume can be 60 cm~3.It was difficult to obtain semi-thin sections of large volume samples.Through analysis of the main process of FFPE,we found that large volume samples were easy to be embrittle when using the traditional FFPE to deal with large volume samples.The optimization mainly focused on the adjustments of clearing and embedding processes.Gradient xylene penetration left tissue less hard and brittle compared with xylene.The adjustment of embedding process and silica gel mold can effectively solve the problem of excessive bubbles in large samples,and silica gel mold can solve the problem of the sag of paraffin blocks.This method retained the advantages of the traditional paraffin-embedding method such as good morphological preservation and sectioning performance.Moreover,it can be used for various large samples such as livers,kidneys and lungs.(2)We develop an improved formalin fixed paraffin-embedding method(i FFPE),which can be used to fluorescence labelled large samples.Firstly,the majority of fluorescence quenching was found to occur during the dehydration steps.Secondly,the types of dehydrating agents were investigated,and it was found that the polarity of dehydrating agents had an important effect on the fluorescent intensity.Thirdly,the effects of dehydrating temperature and the dehydrating temperature on green fluorescent protein stabilization were invested to determine the optimal conditions for fluorescent retention.Meanwhile,the replacement process was adjusted according to the influence of the temperature and time of paraffin-immersion.These adjustments resulted in i FFPE with improved fluorescence preservation.And it was proved that i FFPE can not only preserve the high fluorescent intensity,the fine structures of neurons and long time of fluorescent preservation but also have good compatility with chemical fluorescent tracers.(3)Baesd on i FFPE,we embedded a macaque brain block labeled with rabies virus to trace the input circuits of the superior area 6.We acquired the circuit information at0.65×0.65×10μm~3 resolution with the imaging system for large-volume samples.Furthermore,by image pre-processing and reconstruction of the data set,the input of neurons in superior area 6 was demondtrated at the occipital lobe scale.Then we made a preliminary analysis of neural circuits.In summary,we revealed the mechanism underlying the quench of fluorescence of GFP during paraffin embedding,and developed an optimized protocol.This method not only remained the advantages such as good morphological preservation and sectioning performance but also overcame the limitations of the volume of the specimens and the retention of fluorescent proteins.Therefore,it provides a valuable tool to acquire the structure information of large volume samples in continuous 3D space,such as to study the organization pattern of neural circuits in non-human primates.