Journal: Nature protocols

Article Title: Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

doi: 10.1038/nprot.2015.122

Figure Lengend Snippet: Assembling and working with the PARS chamber. (a) A completed PARS chamber used for whole-body tissue clearing. (b) Individual parts to build a PARS chamber: (1) three 1/8 × 1/8-inch barbed connectors, (2) two 3/32-inch barbed male Luers with locking nut, (3) a 1,000 μl pipette tip box, (4) a 1-gallon Ziploc freezer bag, (5) a three-way stopcock with Luer lock, (6) a 3/32-inch barbed female Luer with full tread, (7) a roll of lab tape, (8) a 22-G × 1-inch gavage needle, (9) a 1/8-inch barbed male slip Luer, (10) a female Luer tee with locks, (11) clay and (12) Tygon E-lab tubing. Ruler shown is 5 cm in length. (c) Three 1/8-inch holes are drilled into the pipette tip box: two into the box front and one into its side, all ~2 cm below the top rim of the box. The three 1/8 × 1/8-inch barbed connectors are placed into the drilled holes. To connect the outflow line (blue tape bands on outflow line tubing), a piece of Tygon tubing is connected from the bottom inside of the pipette box to the single 1/8-inch barbed connector that was inserted through the box side. (d) To continue the outflow line, a second, longer piece of blue-taped tubing is attached to the outer fitting of this same barbed connector (on the outside of the pipette tip box side), and then the other end of this tubing is threaded through the peristaltic pump, pulled back over toward the pipette box and finally connected to a three-way stopcock with a 3/32-inch barbed male Luer with locking nut (rightmost blue-banded tubing in d). To form the inflow line, a short length of tubing (green tape band) is used to connect the three-way stopcock to the front right 1/8-inch barbed connector of the pipette box. The solute flushing line and nitrogen bubbling line, which are subserved by the same tubing (white tape band), are formed by another short length of tubing that joins the third port of the stopcock to the front left 1/8-inch barbed connector. (e) The inflow line is continued inside the pipette box, with the tubing coiled several times around the base of the box so that the solute will be reheated before it passes through the feeding gavage into the subject. The solute flushing line and nitrogen bubbling line is continued inside the pipette tip box and taped to the bottom of the chamber (not shown). (f) The tip of the coiled inflow line tubing is threaded up through the tip wafer (see bird's-eye view of threaded wafer in a) and connected to a 22-G ×1-inch gavage needle with a 1/8-inch barbed male slip Luer. The gavage needle is secured with a short loop of Tygon tubing (~90 mm) threaded through two holes of the wafer. (g) During the polymerization step, the chamber is placed into a 37 °C water bath and sealed in a Ziploc bag. The tubing is attached to the chamber with three 1/8 × 1/8-inch barbed connectors punctured through the Ziploc bag. The Tygon tubing is reconnected from the outside of the bag and surrounded with clay to make an airtight seal. (h) The animal is placed onto the pipette tip box, and the 22-G × 1-inch gavage needle is used to catheterize the heart. (i) The chamber is placed into a 37 °C water bath. A female Luer tee, which is taped onto the lid of the pipette tip box, is punctured through the Ziploc bag, and this joint is sealed with clay to ensure an airtight seal. Finally, to accelerate polymerization, a vacuum line is connected to the female Luer tee to remove oxygen (orange arrow), and a nitrogen gas line (white arrow) is connected to the 1/8-inch barbed connector to deliver a steady flow of nitrogen into the bagged system. The solute is continually circulated through the animal from the outflow line (blue arrow, which also indicates the direction of flow through blue-taped tubing) and inflow line (green arrow, which also indicates the direction of flow through green-taped tubing).

Article Snippet: : 89404-000) Three-way stopcock with Luer lock (World Precision Instruments, cat. no. 14035-10) Luer-to-tubing coupler kit (World Precision Instruments, cat. no. 500895) Barbed fitting assortment kit (World Precision Instruments, cat. no. 500890) 22-G × 1-inch gavage needle (e.g., 22-G, 1.25-mm-tip-diameter straight feeding needle; Fine Science Tools, cat. no 18061-22; Braintree Scientific, cat. no. N-PK 002) Pipette tip boxes; we use empty 1,000-μl racked filter tip boxes (USA Scientific) Optional: 20-G blunt needle (BD Biosciences, cat. no. 305183) and tubing (PlasticsOne) for PARS-CSF 18 C & B Metabond (Parkell, cat. no. S380) Tape (any) Modeling clay (e.g., Sargent Art, cat. no. 22-4400) Peristaltic pump or circulator (e.g., Cole Palmer Masterflex L/S, cat. no. 77800-60; or Cole-Palmer Masterflex L/S Easy Load II head and pump drive, cat. nos.

Techniques: Transferring