Protocols
Precision-Cut Liver Slices Protocols
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√ Rationale for procedural step
♠ Tips & Tricks


  1. Prepare Krebs-Henseleit Buffer (KHB) for slicing and storing precision-cut liver slices.
    1. √ Here’s the simple recipe for KHB:
                   NaCl: 5 mM
                   KCl: 118 mM
                   MgSO4·7H2O: 1.1 mM
                   KH2PO4: 1.2 mM
                   NaHCO3: 25 mM
                   CaCl2·2H2O: 2.5 mM
                   D-Glucose: 25 mM
                   HEPES: 9 mM

  1. Prepare Williams medium E (WME, containing L-glutamine)
    1. √ Here’s the simple recipe for WME:
      WME + L-glutamine: 5 mM
      D-Glucose: 14 mM
      Gentamycin: 50 µg/ml
      Amphotericin B: 2.5 µg/ml


    2. √ Amphotericin B is added to inhibit growth of fungi and yeasts.
  2. Fill the culture plates you will use with the incubation medium.
  3. Warm and oxygenate culture plates with 95% O2/5% CO2 at 37 C for at least 30 min.
  4. Prepare and keep warm a 2-3% solution of agarose at 37 C.
  5. To obtain liver tissue, anesthetize the animal and surgically isolate the liver as quickly as possible, ideally under 5 minutes.
  6. Remove any fatty tissue and transfer cleaned pieces of liver tissue to ice-cold oxygenated KHB. Section out specific liver lobes you need for experiments.  
    1. √  Liver tissue is very sensitive to ischemia, so the time taken for dissection and incubation of precision-cut liver tissue should be as fast as possible. This is especially important for studies of liver metabolism.
  7. Glue the selected liver sample onto the Compresstome® specimen syringe plunger. Place the embedding cap onto the specimen syringe, over the liver sample.
  8. Fill the syringe with 2% agarose (Sigma A-0701, low gelling point, incubated at ~37C).
    1. ​ Order a Starter Kit or additional agarose or blades
  9. ​Draw the syringe downward to bring the liver tissue core sample into the syringe.
  10. Cool the entire contents of the specimen syringe with the chilling block. The liver tissue is now embedded in agarose. The agarose will solidify enough for stable sectioning.
  11. Load the specimen syringe onto the Compresstome® slicer.
  12. The protocol is complete for preparing the liver specimen for sectioning. Proceed from here with normal Compresstome® sectioning procedures.
    1. ♠What are the optimal settings on the Compresstome® for cutting live liver slices? Try a speed (Advance) of 4-5 and an oscillation of 5-7.
Collect each liver slice and immediately transfer to pre-warmed Williams’ Medium E (see recipe above), incubating at 37 C to restore ATP levels and wash away cellular debris.

References

  1. de Graaf IA, Olinga P, de Jager MH, Merema MT, de Kanter R, van de Kerkhof EG, Groothuis GM.  Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. Nat Protoc. 2010 Sep;5(9):1540-51.
  2. Lerche-Langrand C, Toutain HJ. Precision-cut liver slices: characteristics and use for in vitro pharmaco-toxicology. Toxicology. 2000 Nov 16;153(1-3):221-53.
  3. Koch A, Saran S, Tran DD, Klebba-Färber S, Thiesler H, Sewald K, Schindler S, Braun A, Klopfleisch R, Tamura T. Murine precision-cut liver slices (PCLS): a new tool for studying tumor microenvironments and cell signaling ex vivo. Cell Commun Signal. 2014 Nov 7;12:73.
  4. Abdelaal HM, Kim HO, Wagstaff R, Sawahata R, Southern PJ, Skinner PJ. Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining. Biol Proced Online. 2015 Jan 7;17(1):2.

  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