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Optical Clearing Methods for Large Scale Studies of Renal Morphology
Author(s) -
UnnersjöJess David,
Scott Lena,
Blom Hans,
Brismar Hjalmar
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.632.1
Subject(s) - light sheet fluorescence microscopy , immunostaining , microscopy , clearance , autofluorescence , optical sectioning , pathology , confocal microscopy , immunofluorescence , chemistry , biomedical engineering , optics , fluorescence , biology , microbiology and biotechnology , medicine , immunohistochemistry , antibody , physics , scanning confocal electron microscopy , immunology , urology
Due to the limited permeability of visible photons in tissue, renal morphology studies with sub‐cellular resolution have been restricted to mechanical sectioning methods to reconstruct a global view of the kidney. Recent studies on brain tissue have introduced clearing methods that reduce the light scattering in tissue and increase the penetration depth of light. We applied a combination of the CLARITY and SeeDB clearing methods followed by fluorescent immunostaining to obtain optical 3D views of mm‐scale rat kidney samples. Immunostaining was performed using antibodies against protein markers for podocytes (WT‐1 and podocin), blood vessels (aminopeptidase P) and kindey tubules (E‐cadherin). We performed multi‐color fluorescence imaging of stained samples using confocal microscopy. In order to speed up the imaging process and also to some extent improve axial resolution we further applied one‐color light sheet microscopy on cleared samples. Our results show that clearing methods combined with immunostaining is a fruitful tool in studying renal morphology and protein expression with a deep 3D perspective. We present 3D‐renderings of cleared tissue using tubuli‐, glomeruli‐ and blood vessel‐specific markers. By comparing to non‐cleared tissue, we show a substantial decrease in attenuation of light upon clearing, as well as an apparent increase in image quality as function of depth (See figure for cleared 1mm kidney slice). Our observations clearly indicate that light scattering effects in cleared tissue is reduced, which allows for imaging of samples at least an order of magnitude deeper than previously possible using confocal immunofluorescence microscopy at visible wavelengths.