Table 3 Troubleshooting of the deep imaging protocol
From: Deep imaging of LepR+ stromal cells in optically cleared murine bone hemisections
Stepa | Problem | Possible reason | Solution |
|---|---|---|---|
12 | Bone samples crack or break | Decalcification may be needed for longer duration before dehydration for large bone samples such as vertebrae | Remove the surrounding muscles completely during dissection and increase the EDTA incubation time |
12 | Position of the bone samples when sectioning differs across samples | The bone samples were inconsistently oriented in OCT | Always embed the bone samples (step 8) in the same orientation |
22 | A milky suspension is seen upon transfer to BABB | Dehydration was insufficient | Extend the incubation time in methanol (step 21) |
22 | The organ is not sufficiently clear | Incubation in BABB was insufficient | Increase the delipidation time and number of washes with methanol or BABB incubation time |
24 | Air bubbles are present in the mounting system | BABB filling occurred too quickly | Use pipette tip to remove the air bubbles |
25 | The marrow surface is rough | Fixation was inadequate | Increase the incubation time in 4% PFA |
Decalcification was insufficient | Remove the surrounding muscles completely and increase the EDTA incubation time | ||
25 | The staining is uneven | Fixation was inadequate | Increase the incubation time in 4% PFA |
Decalcification was insufficient | Remove the surrounding muscles completely and increase the EDTA incubation time | ||
25 | There is non-specific antibody staining (especially with anti-GFP primary antibody) | Blocking was insufficient | Extend the blocking incubation time or add 0.3 M glycine to the blocking solution |
25 | Non-specific fluorescent crystals are present | Precipitates were present in the antibody solution | Avoid freeze-thaw cycles for the antibodies; spin the secondary antibody before use and use only the supernatant |
25 | Antibody staining is weak | The antibody concentration was too low | Increase the antibody concentration |
There was insufficient penetration of antibodies | Increase the incubation temperature or incubation time for all steps or consider adding up to 1% IGEPAL to the staining solution | ||
25 | Surface antibody staining is strong but inner tissue is weakly stained | The antibody concentration was too high | Decrease the antibody concentration |
25 | Background staining is high | The antibody concentration was too high | Decrease the antibody concentration |
The washing steps after antibody incubation were inadequate | Increase the washing temperature, total washing time, and/or the number of washes |
