1. Volume 93, Issue 4, Pages 1008-1013 (April 2018)
Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion 
David Unnersjö-Jess, Lena Scott, Sonia Zambrano Sevilla, Jaakko Patrakka, Hans Blom, Hjalmar Brismar 
Kidney International 
Volume 93, Issue 4, Pages (April 2018)
DOI: /j.kint
Copyright © 2017 International Society of Nephrology Terms and Conditions

2. Figure 1
Expansion of kidney samples using the magnified analysis of the proteome protocol. (a) A 1-mm-thick piece of adult rat kidney tissue, after polymerization and removal of the excess hydrogel, but before expansion. (b) The same piece of tissue after full expansion protocol. (c) A 1-mm-thick piece of adult rat kidney tissue, after polymerization and removal of the excess hydrogel, but before optical clearing. (d) The same piece of tissue after full clearing protocol. (e) Expansion factors for both cleared and expanded samples. The expansion factors were calculated as the mean linear expansion (width after expansion divided by the same width before expansion) of 3 different samples measured in 2 orthogonal directions. Error bars show SD.
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

3. Figure 2
Comparison of nonexpanded (optically cleared) kidney samples and expanded kidney samples. All samples were stained for podocin with Atto-594 and imaged using a Leica SP8 3X confocal/stimulated emission depletion (STED) microscope (Mannheim, Germany) with a ×100 NA 1.4 oil objective. All images were deconvolved using SVI Huygen software (Hilversum, the Netherlands). (a,b) Images of a nonexpanded (optically cleared) kidney in confocal mode and in STED mode. The pixel size was corrected by dividing with the expansion factor (1.3). Bar = 1 μm. (c) Expanded kidney sample imaged in confocal mode. The pixel size was corrected by dividing with the expansion factor (5). Bar = 1 μm. (d) Schematic drawing of a foot process stained for podocin, and a description of how foot process width and podocin-podocin distance were measured. (e) Mean foot process width measured both in nonexpanded samples imaged with STED microscopy and in expanded samples imaged with confocal microscopy. The mean foot process width measured the distance between 2 slit diaphragms (stained with podocin) in least 30 different positions in 2 different samples. Error bars show SD. (f) Mean podocin-podocin width measured both in nonexpanded samples imaged with STED microscopy and in expanded samples imaged with confocal microscopy. The mean podocin-podocin width measured the distance between 2 podocin strands in at least 30 different positions in 2 different samples. Error bars show SD. (g) The mean foot process width normalized by the mean podocin-podocin width, showing that relative distances in the tissue is preserved following the expansion protocol. Error bars show SD. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

4. Figure 3
Volumetric and multiplex confocal imaging in expanded kidney tissue. All images were acquired using Zeiss LSM780 microscope with a ×40 NA 1.2 water objective. The pixel size was in all images adjusted by dividing with the expansion factor (5). (a) A 20-μm-thick (i.e., 100-μm nonadjusted) confocal stack of a podocin-cre-tdTomato mouse kidney stained for tdTomato with Alexa-488 and podocin with Alexa-555. Bar = 5 μm. (b) Confocal image of expanded podocin-cre-tdTomato mouse kidney stained for tdTomato with Alexa-488 and collagen IV with Atto-594, showing foot processes and the glomerular basement membrane on glomerular capillaries. Bar = 2 μm. (c) Zoom of the marked area in b showing that adjacent foot processes and the glomerular basement membrane are well resolved using confocal microscopy. Bar = 0.5 μm. (d) Confocal image of expanded podocin-cre-tdTomato mouse kidney stained for tdTomato with Alexa-488 and podocin with Alexa555, showing expected localization at the basal part of podocyte foot processes. Bar = 2 μm. (e) Zoom of the marked area in (d) showing the expected 2-sided expression of podocin in the foot processes. Bar = 0.5 μm. (f) Confocal images of an expanded rat kidney stained for podocin with Atto-594 and nephrin with Abberior STAR-635P in adjacent foot processes, showing their expected localizations in the slit diaphragm. Bar = 0.2 μm. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

5. Figure 4
Glomerular nanoscale pathology in expanded kidney samples. All images were acquired using a Zeiss LSM 780 microscope with ×40 NA 1.2 water objective. The pixel size was in all images adjusted by dividing with the expansion factor (5). (a) A 10-μm-thick (i.e., 50-μm nonadjusted) confocal stack showing a glomerular capillary in a podocin-cre-tdTomato mouse kidney stained for podocin with Alexa-555. Bar = 2 μm. (b) A 10-μm-thick (i.e., 50-μm nonadjusted) confocal stack showing a glomerular capillary in a podocin-cre-tdTomato mouse kidney with induced anti–glomerular basement membrane (anti-GBM) nephropathy stained for podocin with Alexa-555, showing effacement of podocyte foot processes. Bar = 2 μm. (c) Confocal image of expanded podocin-cre-tdTomato mouse kidney stained for tdTomato with Alexa-488, showing healthy podocyte foot processes on glomerular capillaries. Bar = 2 μm. (d,e) Confocal images of expanded podocin-cre-tdTomato mouse kidney with induced anti-GBM glomerulonephritis stained for tdTomato with Alexa-488, showing both seemingly healthy foot processes as well as areas with severe effacement. Bar = 2 μm. (f–h) Zoom of the areas marked in (c–e), showing effaced foot processes in mice with glomerulonephritis. Bar = 0.5 μm. (i) Quantification of the degree of effacement in mice with induced glomerulonephritis. The effacement fraction was calculated as the distance where clear foot processes could be visualized, divided by the cross-section length of the capillary wall. The quantification was carried out in 6 different locations in 2 different samples. Error bars show SD. (j) Quantifications of the resolvable number of slit diaphragms found per μm of capillary cross-section length. The quantification was carried out in 6 different locations in 2 different samples. Error bars show SD. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions