Protocols
In Situ Hybridization Protocols
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Key to reading the protocol:
√ Rationale for procedural step
♠ Tips & Tricks

  1. For in-situ hybridization (ISH), fresh tissue should be fixed in 4% paraformaldehyde overnight, then rinsed in 1X PBS the next day. Tissue should be sectioned with the Compresstome® for desired slices.
    • √  Be sure to not “over-fix” tissue in paraformaldehyde! Doing so results in excessive cross-linking of proteins that can interfere with getting good signals for in-situ hybridization.
    • ♠  What slice thickness should you aim for? For ISH, slices should be 3 µm to 7 µm thick . They can be as thick as 10 µm.  
  2. Mount free-floating slices onto Superfrost-Plus slides (Fisher Scientific).
    • √  Why use Superfrost-PLUS slides? Because there is a (+) charge on these types of slides and allow tissue slices to adhere to the slide surface. Therefore, when you are processing the slides, the slices won’t just fall off.
  3. Dry slices on slide using a slide warmer for 30 min at 37°C.
  4. Fix slides again in 4% paraformaldehy in diethyl pyrocarbonate (DEPC) H2O for 20 min.
  5. Rinse slides with DEPC PBS (2 x 5 min).
  6. Deproteinate slices with proteinase K treatment (50 µg.ml; 1M Tris HCl at pH 7.5, and 0.1M EDTA) for 30 min at 25°C.
  7. After deproteination, rinse sections in DEPC PBS (2 x 5 min).
  8. Fixed slides in 4% paraformaldehyde in DEPC H2O for 20 min.
  9. Briefly rinse slides in DEPC H2O.
  10. Next, hybridize slices in hybridization buffer containing the oligonucleotides you are using. Incubate for 24 hours at 37°C in a humid chamber.
  11. After hybridization, wash sections in 0.2X SSC (4 X 20 min each) at 47°C.
  12. Block slices in blocking protein in Tris buffered saline (TBS) for 30 min (Perkin Elmer Amplification Kit).
  13. Incubate slides in digitoxigenin-horseradish peroxidase in TBS containing Tween 20 (TBST; 8 µl/ml) for 30 min at 25°C.
  14. Rinse slides in TBST (3 X 5 min).
  15. Incubate slides in tyramide solution (1:50 in kit diluent) for 10 min at 25°C.
  16. Rinse slides in TBST (3 X 5 min).
  17. Incubate slides in streptavidin-F (1:50) in TNB buffer (0.05 g/ml block protein in Tris-saline) for 30 min.
  18. Rinse slides in TBST (3 X 5 min).
  19. Air dry slides and mount coverslips using Vectashield Mounting Media.

References
* Uses the Compresstome® for successful in-situ hybridization experiments.

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.

Duncan J, Kersigo J, Gray B, Fritzsch B. Combining lipophilic dye, in situ hybridization, immunohistochemistry, and histology. J Vis Exp. 2011 Mar 17;(49).

Li S, Folkvord JM, Rakasz EG, Abdelaal HM, Wagstaff RK, Kovacs KJ, Kim HO, Sawahata R, MaWhinney S, Masopust D, Connick E, Skinner PJ. Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo. J Virol. 2016 Nov 28;90(24):11168-11180.

Lu Y, Ye JH. Glycine-activated chloride currents of neurons freshly isolated from the prefrontal cortex of young rats. Brain Res. 2011 Jun 1;1393:17-22.

Peça J, Feliciano C, Ting JT, Wang W, Wells MF, Venkatraman TN, Lascola CD, Fu Z, Feng G. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature. 2011 Apr 28;472(7344):437-42.

Selever J, Kong JQ, Arenkiel BR. A rapid approach to high-resolution fluorescence imaging in semi-thick brain slices. J Vis Exp. 2011 Jul 26;(53).

Vega-Zuniga T, Marín G, González-Cabrera C, Planitscher E, Hartmann A, Marks V, Mpodozis J, Luksch H. Microconnectomics of the pretectum and ventral thalamus in the chicken (Gallus gallus). J Comp Neurol. 2016 Aug 1;524(11):2208-29.
  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
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    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
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    Dr. Perez-Zoghbi, Texas Tech University
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    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