Table 1 Procedure for deep imaging optically cleared mouse bone hemisections
From: Deep imaging of LepR+ stromal cells in optically cleared murine bone hemisections
Step | Procedure |
|---|---|
Sample preparation (timing: 1 h) | |
1 | Dissect femurs and tibias by carefully removing all surrounding tissues. |
Fixation, decalcification, dehydration, and cryoprotection (timing: 1.5 days) | |
2 | Fix the bones with ice-cold 4% PFA for 15 min and incubate at 4 °C on a rotator for 6 h. |
3 | Wash the bones twice with PBS. |
4 | Incubate the bones with EDTA under constant agitation on a rotator at 4 °C for 12 h. |
5 | Wash the bones twice with PBS. |
6 | Incubate the bones in ice-cold 30% sucrose solution under constant agitation on a rotator at 4°C for 12 h. |
7 | Remove the sucrose solution from the bones and proceed to OCT embedding. |
Preparing hemisections (timing: 2 h) | |
8 | Embed the bones by carefully placing it in OCT. |
9 | Freeze the samples on flat dry-ice to allow the OCT to solidify completely. |
10 | Place the frozen tissue mold in a pre-cooled (–20 °C) cryostat for 30 min–1 h before sectioning. |
11 | Glue the block to the holder using OCT at –20 °C. |
12 | Cut the long bones until the sinus and the full length of the long bone are fully exposed. |
Immunostaining (timing: 8 days) | |
13 | Transfer the hemisections to 1.5-mL microcentrifuge tubes, place them on ice, respectively. Wash the hemisections twice with 1.5 mL of PBS to remove the OCT completely. |
14 | Discard the PBS and block the hemisections with 5% donkey serum in 0.5% PBST for 6 h at room temperature with shaking. |
15 | Transfer the hemisections to 0.6-mL microcentrifuge tubes with freshly prepared primary antibody solution respectively and incubate at room temperature with shaking for 3 days. |
16 | Transfer the hemisections to new 1.5-mL centrifuge tubes respectively and wash with PBS three times for 15 min each at room temperature. Then wash with PBS with shaking at room temperature overnight. |
17 | Transfer the hemisections to 0.6-mL microcentrifuge tubes with freshly prepared secondary antibody solution respectively and incubate at room temperature with shaking for 3 days in the dark. |
18 | Perform PBS washes as in Step 16. |
Optical clearing (timing: 2 days) | |
19 | One day before optical clearing, prepare the BABB buffer. Incubate the mixture with shaking for at least 24 h. |
20 | Spin down the BABB mixture at 800×g for 15 min at room temperature. |
21 | Dehydrate hemisections with at least three 5-minute washes with 1.5 mL methanol. |
22 | Remove methanol buffer from the tube and replace with 1.5 mL BABB. Incubate the hemisections in BABB with shaking at room temperature in the dark for several hours to overnight. Repeat this step if necessary with fresh BABB for optimal clearing. |
23 | Remove the used BABB and fill the microcentrifuge tube with fresh BABB. Store the hemisections at 4°C in BABB in the dark for up to at least 3 months. |
Preparing mounting dish (timing: 1 h) | |
24 | Squeeze a small amount of silicone onto the center of quartz mold. Place the hemisection on top of the fresh silicone. Make sure the hemisection is oriented with the marrow cavity facing up. Add a few drops of BABB to the hemisection and silicone. Let the silicone foundation solidify at room temperature for 30 min before filling the mold with fresh BABB to immerse the hemisection. |
Image acquisition and processing (timing: 2 days) | |
25 | Use a resonant confocal laser-scanning microscope with z-stack scanning and tiling (e.g., Leica Stellaris or Leica SP8) to obtain sequential depth images. |
26 | Convert the tiled z-stack images into “.ims” or “.aivia.tif” format using Imaris x64 10.1.0 (Bitplane) or Aivia 12.1.0 respectively, followed by three-dimensional rendering, snapshot generation, and quantification. |
