Sample preparation and imaging for large scale 3D spectral confocal imaging of tissues v1
Authors: Ashkar Tarek 
Published: July 2023

Frozen participant samples are first sized and preserved for high-resolution 3D imaging. Preserved biopsy sections are imaged, without staining, to understand the sample’s structure and condition. Next, the biopsy sections are stained with antibodies and fluorescent dyes to identify specific structures and cells with pathological significance. This stained sample is imaged in its entirety giving a 3D image. Finally, the 3D image is analyzed by experts to assess known and novel pathologies. This protocol summarizes both label-free imaging and labeling for fluorescence imaging including the labeling, mounting and imaging approaches. The fluorescent labels used herein include conjugated or fluorescent small molecules and conjugated antibodies. Protocol 2 uses both directly conjugated primary antibodies for identifying specific antigens and indirect labeling with a fluorophore conjugated secondary antibody that recognizes the primary antibody. Multiple rounds of imaging are enabled by the use of a non-hardening mounting media, low charge slides and removable rubber cement as a sealant. Details are described below. Two imaging approaches are outlined: protocol 1 and 2. Protocol 1 involves label free imaging of auto-fluorescent species and second harmonic generation (SHG) with multiphoton excitation. These modalities are followed by protocol 2, which involves staining with fluorescent small molecules and immuno-fluorescence and subsequent large scale 3D confocal fluorescence imaging. There are three different staining strategies used in protocols, summarized in Table 1. These protocols start with fixed 50 µm tissue sections prepared as described in the Cryopreservation protocol. Version 3.0 Structure/cell type Target Species Vendor (cat #) Dilution [stock] Direct labeling Indirect labeling Nuclei DAPI - ThermoFisher (D1306) 1:200 [1 mg/mL] - - vasculature Phalloidin - ThermoFisher O7466 1:200 [2 U/mL] Oregon Green - glomeruli " - " " " - brush borders " - " " " - TAL THP Sheep R&D Systems (AF5144) 1:1000 [0.2 mg/mL] Alexa 546 - Proximal tubules AQP1 Goat Santa Cruz (sc-9878) 1:50 [0.2 mg/mL] - donkey anti-goat Alexa 568 (1:200) Neutrophils MPO Rabbit Abcam (ab9535) 1:50 - donkey anti-rabbit DyLight 594 (1:200) macrophages CD68 Mouse Dako (M0876) 1:50 [0.3 mg/mL] - donkey anti-mouse Alexa 633 (1:200) T-cells CD3 Mouse BD Pharmingen (557706) 1:50 Alexa 647 - vasculature CD31 Mouse NovusBio (NB600-562ss) 1:50 [1 mg/mL] Alexa 660 - Version 4.0 Structure/cell type Target Species Vendor (cat #) Dilution [stock] Direct labeling Indirect labeling Nuclei DAPI - ThermoFisher (D1306) 1:200 [1 mg/mL] - - vasculature Phalloidin - ThermoFisher O7466 1:200 [2 U/mL] Oregon Green - glomeruli " - " " " - brush borders " - " " " - TAL THP Sheep R&D Systems (AF5144) 1:1000 [0.2 mg/mL] Alexa 546 - proximal tubules AQP1 Goat Santa Cruz (sc-9878) 1:50 [0.2 mg/mL] - donkey anti-goat Alexa 568 (1:200) neutrophils MPO Rabbit Abcam (ab9535) 1:50 - donkey anti-rabbit DyLight 594 (1:200) macrophages CD68 Mouse Dako (M0876) 1:50 [0.3 mg/mL] - donkey anti-mouse Alexa 633 (1:200) most hematopoietic cells CD45 Mouse Biolegend (304056) 1:50 [0.5 mg/mL] Alexa 647 - vasculature CD31 Mouse NovusBio (NB600-562ss) 1:50 [1 mg/mL] Alexa 660 -