Tissue Clearing and Deep Imaging of the Kidney Using Confocal and Two-Photon Microscopy
Microscopic and macroscopic evaluation of biological tissues in three dimensions is becoming increasingly popular. This trend is coincident with the emergence of numerous tissue clearing strategies, and advancements in confocal and two-photon microscopy, enabling the study of intact organs and systems down to cellular and sub-cellular resolution. In this chapter, we describe a wholemount immunofluorescence technique for labeling structures in renal tissue. This technique combined with solvent-based tissue clearing and confocal imaging, with or without two-photon excitation, provides greater structural information than traditional sectioning and staining alone. Given the addition of paraffin embedding to our method, this hybrid protocol offers a powerful approach to combine confocal or two-photon findings with histological and further immunofluorescent analysis within the same tissue.
Key wordsConfocal Imaging Kidney Three-dimensional Tissue clearing Two-photon
The authors would like to thank Drs. Karen Price, Maria Kolatsi-Joannou, Sarah Ivins, and Catherine Roberts and the rest of the Kidney Disease and Development Group and the Molecular Medicine Unit at UCL Great Ormond Street Institute of Child Health for their guidance, and the staff of the UCL Confocal Imaging Facility for their advice. We would also like to acknowledge Dr. Nicolas Renier (Institut du Cerveau et de la Moelle Epiniere), Dr. Alexander Combes, and Professor Melissa Little (Murdoch Children’s Research Institute) for their email correspondence and clarifications of the methods used in their publications. DJ is supported by funding from the Child Health Research PhD Studentship at UCL Great Ormond Street Institute of Child Health and the UCL MB/PhD Programme. DAL’s laboratory is supported by a Medical Research Council New Investigator Award (MR/J003638/1) and project grant (MR/P018629/1), project grants from Diabetes UK (13/0004763, 15/0005283), and Kidney Research UK (RP36/2015), and by the National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) at Great Ormond Street Hospital (GOSH) for Children NHS Foundation Trust and University College London. Microscopy was performed at the Light Microscopy Core Facility at UCL Great Ormond Street Institute of Child Health, also supported by the NIHR BRC at GOSH (17DD08).
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