Protocols
Precision-Cut Lung Slices Protocols
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Key to reading the protocol:
√ Rationale for procedural step
♠ Tips & Tricks

  1. Anesthetize the animal, then dissect to cannulate the exposed trachea with a catheter.
  2. Cool the live lung specimen with 4°C saline.
  3. (Optional) Inject a bolus of agarose gel (2%) to fill the pulmonary artery.
  4. This process of filling with agarose allow for inflation of alveoli with the same embedding material, and prevents the alveoli from collapsing during the cutting process. Some lung specimens may be sectioned without agarose infusion, especially if the lung specimens are from bovine or sheep animal models (because the quantity of agarose needed for infusion exceeds several liters).
  5. Using a syringe, inject a bolus of air to clear the airway.
  6. Injection of air helps infused agarose reach the alveoli.
  7. Select a section of the lung lobe you would like to take tissues from for sections.
  8. Glue the tissue sample onto the Compresstome® specimen syringe.
  9. Draw the syringe downward to bring the lung tissue core sample into the syringe.
  10. Fill the syringe with 2% agarose (Sigma A-0701, low gelling point, incubated at ~37°C).
  11. Cool the entire contents of the specimen syringe with the chilling block. The lung tissue sample is now embedded in agarose. The agarose will solidify enough for stable sectioning.
  12. Load the specimen syringe onto the Compresstome® slicer.
  13. The protocol is complete for preparing the lung tissue core specimen for sectioning. Proceed from here with normal Compresstome® sectioning procedures.

♠ PCLSs produced can be maintained overnight in Dulbecco’s modified Eagle medium (37°C, 5% CO2) supplemented with 1% penicillin-streptomycin solution.

References

Uses the Compresstome® for successful precision cut lung slices.
  • Graham JG, Winchell CG, Kurten RC, Voth DE. Development of an Ex Vivo Tissue Platform To Study the Human Lung Response to Coxiella burnetii. Infect Immun. 2016 Apr 22;84(5):1438-45.
  • Hiorns JE, Bidan CM, Jensen OE, Gosens R, Kistemaker LE, Fredberg JJ, Butler JP, Krishnan R, Brook BS. Airway and Parenchymal Strains during Bronchoconstriction in the Precision Cut Lung Slice. Front Physiol. 2016 Jul 21;7:309.
  • Oenema TA, Maarsingh H, Smit M, Groothuis GM, Meurs H, Gosens R. Bronchoconstriction Induces TGF-β Release and Airway Remodelling in Guinea Pig Lung Slices. PLoS One. 2013 Jun 26;8(6):e65580.
  • Royce SG, Nold MF, Bui C, Donovan C, Lam M, Lamanna E, Rudloff I, Bourke JE, Nold-Petry CA. Airway Remodeling and Hyperreactivity in a Model of Bronchopulmonary Dysplasia and Their Modulation by IL-1 Receptor Antagonist. Am J Respir Cell Mol Biol. 2016 Dec;55(6):858-868.
  • Yim PD, Gallos G, Perez-Zoghbi JF, Trice J, Zhang Y, Siviski M, Sonett J, Emala CW Sr. Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle. J Smooth Muscle Res. 2013;49:112-24.
  1. "For the past several months, I have been using the Precisionary Instruments Inc VF-200 to cut 300 um prefrontal cortical slices for electrophysiological recordings. As a result, I have been consistently cutting exceptional quality brain slices and have succeeded in obtaining stable whole-cell patch recordings from numerous neurons. Because I am using transgenic mice that have fluorescent reporters, it is of great importance to achieve maximal cell viability and minimal cell death. When I use the VF-200, I have significantly greater number of surface neurons than those slices cut from a regular Vibratome™. In addition the slice surface is much more even, which allows for better imaging. I am thoroughly impressed with the VF-200 and would not hesitate recommending this quality product to fellow electrophysiologists ."
    Newton Woo, PhD NICHD/NI
  2. We obtained a VF-300 Compresstome™ about 5 months ago to use alongside a microtome that we had exclusively used for at least 5 years prior. The VF-300 is more consistent in generating uniform slices and leaves the structure of the tissue/cells more intact than the method we had been using. We have been able to extend the amount of time over which we can cut lung slices, and allows preservation of the architecture of the slice and greater viability of the tissue.
    Dr. Cynthia Kozial-White, University of Pennsylvania
  3. With the Compresstome™ I was able to prepare high quality slices with excellent preservation near the slice surface. The main advantages of this machine are the rapid speed of slicing and the manual stabilization afforded by the agarose embedding. I am able to prepare uniform slices in both the coronal and horizontal planes, and I routinely complete transcardial perfusion, brain removal, and slicing all within less than 10 minutes, which is half the time needed with the Vibratome model. By limiting the time required for slicing the brain tissue is able to recover more easily without significant anoxia, and my slices stay viable for up to 8 hours. I have successfully been able to do targeted whole-cell recordings from fluorescently labeled neurons in slices prepared from >1 year old mice, which is not generally possible with conventional slicers and methods.
    Dr. Jonathan Ting, Allen Institute
  4. We found the Compresstome produces a less apparent damage to the cells of the airways and arterioles as well as to the delicate alveolar parenchyma. Using the Compresstome we are able to obtain more reproducible and successful experiments.
    Dr. Perez-Zoghbi, Texas Tech University
  5. We spent six months trying to cut our tissue (avian olfactory) with a Vibratome..... but without success. It took just 10 minutes with the Compresstome, and the sections are beautiful. It's the best investment my lab has made.
    Dr. David Keays, Institute of Molecular Pathology Vienna
  6. At first, I was skeptical about the VF-200, but the results were astonishing. I have been using a Vibroslice ™ (Campden Instruments, Leicester , UK) for over 13 years, and I would never get so many living cells in one field as with the VF-200. This is also true and especially impressive for tissue of older animals. The oldest rats we used were 31 days of age. The cell quality was very good and the field was very clean. The VF-200 has greatly improved our productivity. Many thanks to Precisionary Instruments Inc. for creating such a wonderful instrument, and for their significant contribution to the neuro-science field.
    Dr. Jiang-Hong Ye, University of Medicine and Dentistry of New Jersey