Fluorescent-Protein Stabilization and High-Resolution Imaging of Cleared, Intact Mouse Brains
Fig 1
Changes in EGFP fluorescence in E.coli during dehydration and clearing.
(A) Fluorescence of fixed, EGFP-expressing E. coli cells during dehydration and clearing. Dehydration steps: 2.5 h each (80%: 16 hours); clearing steps as indicated, all steps at RT, pH unadjusted. (B) Fluorescence of fixed, EGFP-expressing E. coli cells after dehydration in pH-adjusted alcohol solutions as indicated, followed by 5 d clearing at the respective pH (all steps at RT, data from different experiment). (C) LSFM recording (sagittal optical slice) of olfactory bulb EGFP fluorescence of a brain from a two weeks old transgenic TgCamKII-EGFP mouse. After fixation, the two hemispheres of the brain were separated. One hemisphere was dehydrated and cleared according to Dodt et al. (“E-BABB”, left) [4], the other hemisphere using 1-propanol for dehydration and BABB for clearing, all steps at pH 9.5 (“1P-BABB”, right). For both samples, dehydration steps were 8h and 16h alternating; clearing step: 7 hours; all steps at RT. The brightness and contrast settings were set to two different linear ranges (indicated on the left) which were chosen to show the 1P-BABB sample intensity range (upper panels) and the E-BABB sample intensity range (lower panels). gl, glomerular layer; aob, accessory olfactory bulb. Scale bar, 1mm. (D) Fluorescence of fixed, EGFP-expressing E. coli cells after dehydration with 1-propanol, or tert-butanol, and additional five days in clearing solution, all steps at the indicated temperatures, all solutions at pH 9.5.