Four to five millimeter thick hippocampal and amygdaloid slices were cleared based on the protocol described by Ye et al. (2016) (link)). Briefly, >10 weeks old mice were transcardially perfused with ice cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA), brains were removed and kept in 4% PFA overnight at 4°C. Brains were then transferred to a hydrogel solution (PBS with: 2% acrylamide, bio-rad #161-0140; 0.1% Bisacrylamide, bio-rad #161-0142; 0.25% VA-044 initiator, Wako, 011-19365; 4% PFA) for 2 days. The samples were then degassed with N2 for 45 min and polymerized in 37°C for 3.5 hr. The samples were then washed overnight in 200 mM NaOH-Boric buffer (sigma, #B7901) containing 8% sodium dodecyl sulfate (SDS) (sigma, #L3771), to remove PFA residuals. Samples were then stirred in a clearing solution (100 mM Tris-Boric buffer, bio-lab, #002009239100 with 8% SDS) at 37°C for 3–4 weeks. After the samples became transparent, they were washed with PBST (PBS with 0.2% tritonX100; ChemCruz, #sc-29112A) for 24 hr at 37°C with mild shaking and for another 24 hr with fresh PBST 0.2% at RT. Finally, the samples were incubated in the refractive index matched solution Rapiclear (RI = 1.47; SunJin lab, #RC147002) for 10 hr at 37°C and 2 days at room temperature before imaging.
Va 044 initiator
Manufactured by Fujifilm
The VA-044 initiator is a chemical compound used in laboratory settings. It functions as a thermal initiator, capable of generating free radicals when exposed to elevated temperatures. The core purpose of the VA-044 initiator is to facilitate controlled radical polymerization reactions, a commonly utilized technique in various fields of chemical research and development.
Automatically generated - may contain errors
Lab products found in correlation
Saponin, by Merck Group (3 mentions)
Acrylamide bis solution, by Bio-Rad (2 mentions)
Gelslick, by Lonza (2 mentions)
Acrylamide, by Bio-Rad (2 mentions)
Si86 1, by Thermo Fisher Scientific (2 mentions)
Skyscan 1272 scanner, by Bruker (2 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Acrylamide, by Thermo Fisher Scientific (1 mentions)
Bis acrylamide, by Bio-Rad (1 mentions)
7 protocols using va 044 initiator
1
Tissue Clearing of Hippocampal and Amygdaloid Slices
2
Single-Molecule RNA Hydrogel Immobilization
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Coverslips were first sonicated at 100% ethanol for 20 minutes, followed by plasma cleaning with a plasma cleaner at HIGH for 5 minutes. The coverslips were then immersed in the 2% PlusOne bind-silane(17-1330-01) solution made in ethanol for 30 minutes at room temperature. After rinsing the coverslips with ethanol for several times, the coverslips were dried at 90°C for 30 minutes. Purified total RNA was mixed in 4% acrylamide/bis solution (1610147; Bio-Rad) with fresh 25mM VA-044 initiator (27776-21-2; Wako Chemical) and the solution was degassed for 10 minutes on ice. A 12mm square coverslip (470019-000; VWR) was functionalized with GelSlick (Lonza; 50640). 1uL of the RNA hydrogel solution was added to the bind-silane functionalized coverslip and was spread out using the GelSlick functionalized square coverslip. The thickness of the hydrogel formed can be controlled by manipulating the volume added. The polymerization happened in a humid hybridization at 37°C for 2 hours. After polymerization is complete, the coverslips were immersed in 2× SSC for an hour or more to facilitate the removal of the top coverslips. smFISH measurement was then performed according to standard protocol.
3
Hydrogel Embedding for Tissue Fixation
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Murine tissues were fixed in hydrogel monomer solution (HMS) composed of 4% (wt/v) acrylamide, 4% (wt/v) paraformaldehyde, and 0.25% (wt/v) VA-044 Initiator (WAKO, cat. No 017-19362) in PBS. The lumen of entire gut segments was perfused with HMS before the specimen was submerged in HMS for 2 days at 4 °C.
After fixation, individual specimens were put into 50 ml tubes with sufficient HMS to cover them entirely. The tubes were placed in a vacuum oven (Thermo Scientific Heraeus, Hanau, Germany) and degassed at 13.3 kPa for 15 min. Afterwards, the oven was heated to 37 °C for 3 h at 13.3 kPa to start polymerization. After the gel solidified, we used paper tissues to gently remove gel rests from the tissue.
After fixation, individual specimens were put into 50 ml tubes with sufficient HMS to cover them entirely. The tubes were placed in a vacuum oven (Thermo Scientific Heraeus, Hanau, Germany) and degassed at 13.3 kPa for 15 min. Afterwards, the oven was heated to 37 °C for 3 h at 13.3 kPa to start polymerization. After the gel solidified, we used paper tissues to gently remove gel rests from the tissue.
4
Single-Molecule RNA Hydrogel Immobilization
5
3D Embryonic and Skull Imaging
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Embryos were dissected in ice-cold PBS and fixed in 4% PFA overnight at 4 °C. After fixation, embryos were transferred to new tubes and immersed in fresh stabilization buffer [4% PFA, 4% acrylamide (Bio-Rad, #161-0140), 0.05% bis-arcylamide (Bio-Rad, #161-0142), 0.25% VA044 initiator (Wako Chemical, #017-19362), 0.05% Saponin (Sigma, #84510) in PBS] for 3 days at 4 °C. Then, sample tubes were capped off and purged with nitrogen at 10 psi for 3 min. Tubes with samples were recapped and incubated for 3 h. at 37 °C for the hydrogel to polymerize. Samples were removed from the hydrogel and stored in PBS with 0.1% sodium azide at 4 °C until ready for imaging. Before micro-CT scanning, embryos were immersed in 0.1 N iodine (Thermo Fisher, #SI86-1) and rocked overnight at room temperature. Micro-CT images were acquired using a Bruker SkyScan 1272 Scanner at a 11μm resolution. Adult skulls from wild type, Hcfc1A115V/Y, and Hcfc1A115V/Y; Mmachc-OE+/tg mice were similarly prepared, but without iodine immersion.
6
Tissue Fixation and Staining Protocol
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For tissue fixation, either 4% Paraformaldehyde (PFA) or hydrogel (Hg) was utilized. The 4% weight per volume (w/v) PFA was prepared by dissolving powdered PFA (Sigma-Aldrich, 158127) in 0.01M Phosphate Buffered Saline (PBS) at 65°C and was made fresh for perfusion. Hg solution was prepared using a previously published recipe (4% volume per volume (v/v) PFA (Sigma-Aldrich, 158127), 4% (v/v) acrylamide (Fisher Scientific, 79-06-1), 0.05% (v/v) Bis acrylamide (Boston Bioproducts, Bis-2), 0.25% (w/v) VA-044 initiator (Wako, 27776-21-2), 0.05% (w/v) Saponin (Sigma-Aldrich, 8047-15-2), 0.01M PBS, in ddH2O) [12 (link),18 (link)] and was kept frozen until use, and then was thawed at room temperature before use.
Iodine-potassium iodide (I2KI) solution was used to stain the soft tissue because of its quick penetration and low toxicity. Although elemental iodine (I2) does not dissolve in water in any appreciable amount by itself, with the addition of potassium iodide (KI), it forms tri-iodide ions with increased solubility [7 (link)]. Thus, we prepared I2KI stain by dissolving 2% (w/v) elemental iodine (I2) (Acros, 19656) and 4% (w/v) potassium iodide (KI) (Sigma-Aldrich, 746428-800G) in Ultrapure water.
Iodine-potassium iodide (I2KI) solution was used to stain the soft tissue because of its quick penetration and low toxicity. Although elemental iodine (I2) does not dissolve in water in any appreciable amount by itself, with the addition of potassium iodide (KI), it forms tri-iodide ions with increased solubility [7 (link)]. Thus, we prepared I2KI stain by dissolving 2% (w/v) elemental iodine (I2) (Acros, 19656) and 4% (w/v) potassium iodide (KI) (Sigma-Aldrich, 746428-800G) in Ultrapure water.
7
Hydrogel-Embedded Embryo Imaging
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