We found that the entire = 3 slices (scale bar: 2 mm). Glucagon receptor antagonists-1 (C) Analysis of the fluorescence of the background shows that the background is usually unchanged before and after AKS treatment (n = 7 background area). All values are included in Additional file 11: Table S6. (D) Analysis of the fluorescence of the YFP transmission, showing that this fluorescence of YFP was enhanced after AKS treatment (= 7 YFP-labelled neurons). All values are included in Additional file 11: Table S7. Statistical significance (**** 0.0001) was assessed by two-tailed Students t-test. 12915_2022_1275_MOESM1_ESM.jpg (294K) GUID:?FA3A3F67-C765-446A-965A-ACAA1F17C73C Additional file 2:?Physique S2. The fluorescence preservation before and after 10 min, 24 h, 7 days, and 14 days of treatment with AKS. We Glucagon receptor antagonists-1 tested four fluorescent proteins and nine fluorescent dyes, using the same imaging parameters to observe the same location of the slices at all detection time points. We found that AKS treatment for 10 min only reduced the fluorescence of Dylight405, and other fluorescence signals were well preserved. However, after two weeks of AKS treatment, the tdTomato transmission and Retro-Beads transmission also weakened, but imaging could still be performed. Representative of n = 3 slices each conditioning. (Level bar: 50 m). 12915_2022_1275_MOESM2_ESM.jpg (4.3M) GUID:?042FD9F1-7240-4AEF-880D-DA7D9C96B4D6 Additional file 3:?Physique S3. Analysis of the fluorescence quench of ChR2-YFP before and after AKS treatment at the same imaging parameters. (A) Time-stack images showing the photobleaching effect of ChR2-YFP before and after AKS treatment around imaging time at identical laser power. Representative of n = 3 slices (Scale bar: 20 m). (B) Analysis of the relative mean fluorescence intensity around time-series imaging of A (n = 3 slices). All values are included in Additional Glucagon receptor antagonists-1 file 11: Table S8. 12915_2022_1275_MOESM3_ESM.jpg (495K) GUID:?98F7D256-5517-489D-85EF-DB205029BB09 Additional file 4:?Physique S4. 3D imaging of peripheral tissues cleared with AKS using two-photon microscopy. Tissue sections from 3-month-old mouse testis (300-m-thick), intestines, liver (200-m-thick), Glucagon receptor antagonists-1 and kidney (200-m-thick) were permeated in PBS made up of 0.3% Triton X-100 (PBST) for three hours at room temperature, and then washed with PBST for three times, each time for 1 h. After washing, the tissues were stained with a solution made up of DAPI and lectin-Dylight594 (DAPI was used at 1:1000 and lectin-Dylight594 (VECTOR laboratories, DL-1177-1) was used at 1:500 diluted in PBST contain 0.05% NaN3), and staining was processed at 37C for 24 h. Then the sample was washed three times with PBST, each time for one hour. After washing, the tissues were imaged in PBST using two-photon microscopy. We used an 800 nm laser for excitation of DAPI and Dylight594. After the imaging, the tissues were cleared in AKS for 1 h at room temperature, and then imaged with the same imaging parameters used before clearing. We found that AKS treatment increased the imaging depth of all these tissues, which indicated that AKS could be utilized for 3D imaging of peripheral tissues. It should be noticed that after AKS treatment, DAPI was visible in the entire imaging range, but lectin-Dylight594 only appeared on the surface of the liver and kidney slices. We presume that it may be due to our insufficient staining time or insufficient amount of dye, which have resulted in only the surface staining. Representative of n = 3 samples of each tissue. 12915_2022_1275_MOESM4_ESM.jpg (4.5M) GUID:?B90E28E3-E95B-4E1B-9D3D-68DA9A7165F7 Additional file 5:?Physique S5. Analysis of the morphology switch of 300-m-thick human brain slices after 10 min of AKS treatment. (A) Image of three 300-m-thick human brain slices before and after AKS treatment. (B) Analysis of the morphology switch before and after 10 min of AKS treatment (n = 3 slice). The tissue linear growth was 96.33 2.06% (mean SEM) after 10 min AKS treatment and had no significant difference compared with PBS ( 0.05). Statistical significance was assessed by two-tailed Students mice central amygdala. 12915_2022_1275_MOESM7_ESM.mp4 (7.1M) GUID:?BE126D40-3F08-439A-96EE-3E3465A26133 Additional file 8:?Movie S2. 3D visualisations of tdTomato-labelled CRH neurons (reddish) in mice paraventricular nucleus of the hypothalamus. 12915_2022_1275_MOESM8_ESM.mp4 (18M) GUID:?E889995A-405D-455B-89ED-EBCB04F21155 Additional file 9:?Movie S3. 3D visualisation of Thy1-YFP mouse brain slices immunostained for the astrocyte marker. YFP, Mouse monoclonal to TGF beta1 green; astrocyte, reddish. 12915_2022_1275_MOESM9_ESM.mp4 (9.5M) GUID:?098A688A-8EB0-4BA9-9A9D-AFDCD5799B8B Additional file 10:?Movie S4. 3D visualisation of thioflavin-S labelled neuritic plaques (green) and GFAP labelled astrocytes (reddish) in human brain samples with Alzheimers disease. 12915_2022_1275_MOESM10_ESM.mp4 (7.4M) GUID:?1292AC4F-DCFD-4E7A-A707-4B0D8CD104C1 Additional file 11:?The individual raw data values of figures and Additional files for quantity of replicates 6. All the data are cited in the physique story. 12915_2022_1275_MOESM11_ESM.zip (142K) GUID:?10F51BFD-24AB-4F16-B369-79AB21A85E3C Data Availability StatementAll data generated or analysed during this study are included in this published article and its supplementary information files. Abstract Background Tissue-clearing Glucagon receptor antagonists-1 techniques have recently been developed to make tissues transparent for three-dimensional (3D) imaging at different scales, including single-cell.