Transcardiac Perfusion

• You must complete required lab safety training before starting this procedure.

• You must complete required animal use training and listed on IACUC protocol before starting this procedure.

• If this is your first time doing this procedure, ask to be trained by an experienced lab member.  If you have not done this in a while, you should ask for a refresher.

• Also review the following material:

  • Kuwajima M, Mendenhall JM, Harris KM (2013) Large-Volume Reconstruction of Brain Tissue from High-Resolution Serial Section Images Acquired by SEM-Based Scanning Transmission Electron Microscopy. Methods Mol Biol (Nanoimaging: Methods and Protocols) 950:253-73. (PDF)

• Further reading:

  • Hayat MA (1981) Fixation for electron microscopy. Academic Press

  • Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A-138A. (PDF)

  • Tao-Cheng JH et al. (2007) Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain. J Comp Neurol 501:731-740. ()

  • Dehghani A et al. (2018) Nuclear expansion and pore opening are instant signs of neuronal hypoxia and can identify poorly fixed brains. Sci Rep 8:14770. ()

• Before starting, even if you have done this procedure before,

  • read this protocol entirely.

  • If you are using adult rats, review the instruction movie on the tracheotomy procedure.

  • review relevant Safety Data Sheets and Harris Lab SOP (also see below).

  • ensure you have all reagents and supplies listed below.

  • ensure all equipment is in good working order.

    • Isoflurane vaporizer must be serviced annually; check tubing, surgical instruments, etc.

  • have all waste containers ready (also see Clean-up).

  • plan your schedule well so that you wouldn’t be rushed to complete the procedure.

• Review SDS and Harris Lab SOP for the following hazardous chemicals used in this procedure:

  • Ethanol: flammable; irritant (eye)

  • Formaldehyde

  • Glutaraldehyde

  • Hydrochloric acid

  • Isoflurane

  • Sodium cacodylate: carcinogen; irritant (skin, eye); skin permeator

  • Sodium hydroxide

• Other hazards associated with this procedure include:

  • Physical: compressed gas cylinder (O₂/CO₂)

  • Sharps: Surgical scissors, scalpel, needles

  • Biological: Live rats/mice, blood, animal waste and soiled cage bedding

• The following Personal Protective Equipment is required for this procedure:

  • Lab coat

  • Nitrile gloves (double-layer required; regularly check for holes)

  • Eye goggles

  • Mask

  • Face shield

• Place a piece of absorbent sheet on the work surface before starting the procedure.  When done, discard into the “Solid Waste – No UA” bag

• Eye goggles, face shield

• Gloves (nitrile)

• Lab coat

• Mask

• Plastic Apron (optional)

• Barbed fittings from Cole-Parmer

• Luer kits from World Precision Instrument

• Silicon rubber tubing for anesthesia line

• Vacuum line for waste collection system

• IV line with clamp and flow regulator for perfusate lines

  • Perfusate lines should be transparent enough to be able to see air bubbles.

• Tygon tubing for general use

• Sphygmomanometer with a hand pump (cut off the cuff)

  • Use hand pump in the absence of an air pump.

• Air pump: Perfusion Two (Leica Biosystems 39471010)

  • This pump is designed to deliver constant air pressure at 300 mmHg. To be able to adjust pressure, we added a regulator (Air Logic R-7010-2B2B-W/K) between "Air Tank" and "Perfusion Pressure" switches (See Figures 2-4).

• Air filter (Parker Hannifin 9933-05-AAQ)

• 1-L glass bottle (Kimax, or equivalent) × 1

  • This can be substituted by a plastic air tank (e.g., Clippard AVT-PP-35).

• #6 two-hole stopper × 1

• O₂/CO₂(95%/5%) cylinder with regulator and support

• Isoflurane (Animal Health International 19632158; stored at RT in well-ventilated area)

• Medical grade O₂ cylinder with regulator and support (Can be replaced by O₂/CO₂ or an oxygen concentrator)

• Large glass desiccator, wad of 4-5 Kimwipes, Pasteur pipet

• Matrx VIP3000 Isoflurane Vaporizer (Stoelting)

  • This apparatus must be serviced annually.

  • For Central Texas area: William Connery, Handlebar Anesthesia, (512) 423-7668, handlebaranesthesia@yahoo.com

• A small animal ventilator (Harvard Apparatus Model 683, or equivalent)

• Nose cone (e.g., Stoelting; Kent Scientific)

• endotracheal tube (a modified 16-gauge hypodermic needle) with Y-connector

• Optional: Plexiglas anesthesia induction chamber (e.g., Stoelting; Kent Scientific)

• Deep water bath (e.g., VWR Digital Unstirred Water Bath, L×W×D = 12⁷/₈"×11¹³/₁₆"×5²⁹/₃₂"; VWR 89032-216)

• Lead donuts × 2

• #6 stoppers: three-hole × 1, two hole × 1

• 3-way valve × 1

• Flow regulator × 1

• 13-gauge needle with 60° bevel with cork disk ½ inch from tip (to restrict depth of penetration; use a16-gauge for juvenile rats)

• Air stone × 1

• 4-L vacuum flask (preferably plastic-coated) × 1

• #11½ one-hole stopper × 1 

• Vacuum line filter (Whatman VACU-GUARD, VWR 28137-858), 1 per day of perfusions

Scissors and knife etc. should be sharpened periodically.

• Stainless steel dissection tray with Styrofoam board cut to fit snugly lengthwise in the tray with ~1-inch space on one side. 

• Pins to secure animal to Styrofoam board

• Scalpel and blades

• Assorted dissecting scissors

• Forceps

• Hemostats

• Retractors

• Knife (to decapitate rat)

• Bone Rongers

• Spatulas

• Silk suture (or cotton tread from fabric store can be used instead)

• Gauze pads

• Applicator sticks

Open

All reagents are stored at RT unless otherwise noted.

• Calcium chloride dihydrate (CaCl₂·2H₂O; Sigma-Aldrich 223506)

• d-Glucose (Sigma-Aldrich G7528)

• Formaldehyde (20% aqueous solution in 100 ml bottles; Ladd Research 20304)

• Glutaraldehyde (50% aquieous solution in 100 ml bottles; Ladd Research 20211; stored at 4°C)

• Hydrochloric acid (HCl; 1 M aqueous solution; for adjusting pH)

• Magnesium sulfate heptahydrate (MgSO₄·7H₂O; Sigma-Aldrich M5921)

• Potassium chloride (KCl; Sigma-Aldrich P9333)

• Sodium bicarbonate (NaHCO₃; Sigma-Aldrich S6297)

• Sodium cacodylate trihydrate (Ladd Research 20305)

• Sodium carbonate (Na₂CO₃; Sigma-Aldrich 223530)

• Sodium chloride (NaCl; Sigma-Aldrich S7653)

• Sodium hydroxide (NaOH; 1 M aqueous solution; for adjusting pH)

• Purified water

  • double-distilled, ASTM type I, WFI (water for injection), or equivalent; e.g., Fisher 91-502-5

  • ASTM type I water can be made with a properly maintained water filtration system (e.g., Milli-Q® Type 1 Ultrapure Water System )

• 2- or 4-L glass beaker × 2

• Magnetic stirrer × 2

• 100-ml glass beaker and a disposable pipet (for dissolving sodium carbonate [Na₂CO₃])

• 1- or 2-L graduated cylinder (depending on the final volume)

• pH meter (and calibration standards)

• 1- or 2-L glass bottle (Kimax, or equivalent) × 2

• Vacuum filtration system (pore size = 0.22 µm; e.g., VWR 10040-468 – this one screws onto Kimax glass bottles)

• WESCOR VAPRO 5520 Osmometer (with calibration standards, filter paper disks, pipet, and pipet tips)

• Timer (in count-up mode)

• Worksheet