After extraction of the petrous bones, all samples were washed with phosphate buffered saline (PBS) three times for 30 min each, then fixed in 4% paraformaldehyde (PFA) overnight. Bone clearing was carried out using the PEGASOS method [33 (link)]. Briefly, bone samples were immersed in 20% EDTA (pH 7.0) at 37°C in a shaker for 4 days for the mouse petrous bone and 16 days for the pig petrous bone (L3) until complete decalcification was achieved. The mice experiments were pre-approved by the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute. To remove excess EDTA, samples were then washed with distilled water for 30 min. Then, decolorization with 25% w/v Quadrol (Sigma-Aldrich; 122262) for 2 days at 37°C in a shaker. Samples were then placed in tert-butanol (tB) (Sigma-Aldrich; 471712) and 3% w/v Quadrol for delipidation for 2 days with gradual increasing of (tB) concentration (30%, 50% and 70% v/v). Then 70% v/v tert-butanol with 30% v/v polyethylene glycol (Sigma-Aldrich; 447943) (tB-PEG) and 3% w/v Quadrol solution was used for 2 days for dehydration. Samples were then immersed in 75% v/v benzyl-benzoate (Sigma-Aldrich; B6630) with 25% v/v polyethylene glycol (BB-PEG) medium at 37°C for at least 1 day for clearing. All samples were preserved in freshly prepared BB-PEG clearing medium (refractive index R.I. = 1.54) at room temperature in the dark.
Quadrol
Manufactured by Merck Group
Sourced in United States
Quadrol is a multi-purpose laboratory equipment designed for various applications in scientific research and analysis. It functions as a precision analytical balance, providing accurate measurements of sample weights. The Quadrol is a reliable and versatile tool for researchers and laboratory professionals.
Automatically generated - may contain errors
Lab products found in correlation
Peg mma 500, by Merck Group (5 mentions)
Triton x 100, by Merck Group (3 mentions)
Triethanolamine, by Merck Group (3 mentions)
Urea, by Merck Group (2 mentions)
Va 044, by Fujifilm (1 mentions)
Peg1000, by Merck Group (1 mentions)
Pegdma, by Polysciences (1 mentions)
Peg 200, by Merck Group (1 mentions)
Peg400, by Merck Group (1 mentions)
Stereomicroscope, by Zeiss (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Kolliphor el, by Merck Group (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
Histodenz, by Merck Group (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
Urea, by Solarbio (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Ultramicroscope, by Miltenyi Biotec (1 mentions)
Lsm780 inverted confocal microscope, by Zeiss (1 mentions)
19 protocols using quadrol
1
Bone Clearing and Dehydration Protocol
2
PEGASOS Tissue Clearing for Deep Imaging
3
Transparency Optimization of Squid Mantle
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The whole raw squid was obtained and the head, chitin pen, transparent strip, and viscera inside the squid were removed. The outer membrane was peeled off. Deuterium‐depleted water (DDW) was used to clean the mantle several times at 4°C. Next, the mantle was cut to 10 × 10 mm for each piece, followed by cutting into 3 mm thickness and washing with DDW three times. The squid mantle tissues were divided into eight groups: untreated group (Ctrl), decellularized and fixed and transparent group (DFT), decellularized and transparent group (DT), fixed and transparent group (FT), transparent group (T), transparent‐fixed‐decellularized group (TFD), transparent and decellularized group (TD), and decellularized group (D). Eight groups of squid mantles were performed using an immersion method in a shaker at 37°C with a shaking speed of 100 rpm, as shown in Scheme 2 .
Reagent required for 0.5% SDS method: 5 g of sodium dodecyl sulfate (SDS) powder was dissolved in 1000 mL DDW.
Reagents required for CUBIC method: 80% Quadrol: 500 g Quadrol (Sigma‐Aldrich) was subjected to 125 g deionized water. ScaleCUBIC‐1 (also known as reagent‐1): 125 g urea (Solarbio) and 156 g 80% Quadrol were added in 144 g deionized water with heating and stirring, dissolved fully and then cooled to room temperature (RT), then added 75 g of Triton X‐100 (Solarbio), dissolved fully and then cooled to RT.43
Reagent required for 0.5% SDS method: 5 g of sodium dodecyl sulfate (SDS) powder was dissolved in 1000 mL DDW.
Reagents required for CUBIC method: 80% Quadrol: 500 g Quadrol (Sigma‐Aldrich) was subjected to 125 g deionized water. ScaleCUBIC‐1 (also known as reagent‐1): 125 g urea (Solarbio) and 156 g 80% Quadrol were added in 144 g deionized water with heating and stirring, dissolved fully and then cooled to room temperature (RT), then added 75 g of Triton X‐100 (Solarbio), dissolved fully and then cooled to RT.
4
Tissue Clearing with PEGASOS Method
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Tissue clearing with the PEGASOS method was performed as previously described (Jing et al., 2018) . In brief, after perfusion, mouse mandibles were dissected and fixed in 4% PFA. Decalcification was carried out in 10% EDTA at 37 C for 1 week. Decolorization was carried out by shaking samples in 25% Quadrol (Sigma-Aldrich 122262) at 37 C for 2 days. Delipidation was performed in gradient 30%, 50% and 70% tert-butanol aqueous solutions (Sigma-Aldrich 360538) at 37 C. Dehydration treatment was performed with tB-PEG solution composed of 72% tert-butanol, 25% PEGMMA500 (Sigma-Aldrich 409529) and 3% Quadrol. For clearing, samples were immersed the BB-PEG clearing solution composed of 72% benzyl benzoate (Sigma-Aldrich 409529), 25% PEGMMA500 and 3% until complete transparency was achieved in about 24 hours. Samples were stored in the BB-PEG solution at room temperature for imaging. 3-D imaging was performed with a Zeiss upright LSM780 2-photon microscope. 3D image reconstruction was performed with Imaris 9.0.
5
Quadrol Dilution in Distilled H2O
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Quadrol (N,N,N′,N′-Tetrakis(2-hydroxypropyl) ethylenediaminel) (Sigma-Aldrich 122,262) was diluted with distilled H2O to a final concentration of 25% v/v.
6
Whole-mount Immunostaining of IPF and Donor Lungs
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IPF and donor-fixed lung tissue in optical cutting temperature (OCT) blocks were sectioned at 250 µm and cleared via the passive clarity (PACT) protocol, followed by whole-mount antibody stain.30 (link) In short, OCT is removed with PBS, and then the tissue is placed in a cold 4% polyacrylamide (1610140, Bio-Rad) hydrogel with 0.25% photoinitiator (VA-044, Wako) solution overnight. The tissue was polymerised at 37°C for 4 hours, washed with PBS to remove excess hydrogel and then incubated at 37°C overnight in an 8% sodium dodecyl sulfate in PBS solution for permeabilisation. After washing with PBS, a 25% Quadrol (122262-1L, Sigma Aldrich) solution in PBS was added for 16 hours on a 37°C rotator, after which the samples were washed in PBS and are ready for immunostaining. For immunostaining, the PACT-cleared tissue sample was incubated with 4% donkey serum blocking solution at room temperature and then incubated with primary antibodies aSMA (Sigma A5228) and Abca3 (Seven Hills WMAB-17G524) at 4°C for 3 days. Unbound antibody was removed with PBS, followed by fluorescent labelling with secondary antibodies. After washing with PBS, the tissue was mounted in refractive index matching solution (RIMS) solution for second harmonic generation (SHG) imaging on a Nikon FN I upright microscope and analysed using Nikon Elements and Imaris software.
7
Gradient Delipidation using tB and Quadrol
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tB (Sigma-Aldrich, 360538) was diluted with water to prepare gradient delipidation solutions: 30% (v/v), 50% (v/v) and 70% (v/v). 5% (w/v) Quadrol (Sigma-Aldrich, 122262) was added to adjust the pH over 9.5.
8
Dehydration Solution Composition for Preservation
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9
Fluorescence-Preserving BB-PEG Formulation
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BB-PEG was prepared from mixing 75% v/v benzyl benzoate (BB) (Sigma-Aldrich B6630) and 25% v/v PEGMMA500 (Sigma-Aldrich 409529) supplemented with 3% w/v Quadrol (Sigma-Aldrich 122262) together. The fresh medium was a colorless liquid with low viscosity and turned slightly yellow in a week. Other forms of PEGs including PEGMMA200 (Polysciences 16664), PEGDA400 (Polysciences, 01871), PEGDMA (Polysciences, 00096), PEG200 (Sigma-Aldrich 81150), PEG400 (Sigma-Aldrich 807485), and PEG1000 (Sigma-Aldrich 81190) were also used for comparing their fluorescence preservation capabilities.
10
Golgi-Cox and Golgi Staining Tissue Clearing
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After Golgi-Cox and Golgi staining, samples (sections or brain bocks) were clarified using CUBIC protocol. For Golgi-Cox-stained samples, tissue clearing was started after the incubation with 15% Kodak fixative, and for original Golgi-stained samples, after the 0.05% gold chloride for 30–60 min. Samples were washed three times in dH2O for 10 min, followed by incubation in the CUBIC solution: 250 g/l urea (#821527, ICN Biomedicals, USA), 250 g/l Quadrol (#122262, Sigma-Aldrich, Belgium), and 150 g/l Triton X-100 (#RT22140, EMS, USA), sequentially dissolved in dH2O with heating and stirring, and cooled to RT). Golgi-Cox-stained samples were incubated for 10 min - 8 days (see text) in a mixture of equal parts of CUBIC solution and dH2O, at 37 °C in the dark with agitation. Original Golgi-stained samples were submitted to the same CUBIC protocol and incubations continued until the desired level of tissue clearing was achieved, 1–21 days (see text).
Samples were imaged directly in clearing solutions. 3D light microscopy images were taken with a Bioptonics OPT device (Fig.3k,l , and Supplementary Video 2 ) or a Nikon spinning-disk confocal with a CFI Plan Apo 10XC Glyc Objective for Clearing Applications (Fig. 3e,f ). Whole samples were imaged with a Zeiss Stereomicroscope (Fig. 3a–d,g–j ).
Samples were imaged directly in clearing solutions. 3D light microscopy images were taken with a Bioptonics OPT device (Fig.
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