1Department of Biology, University of Iowa, 2Molecular Targeting Technologies, Inc.
Duncan, J., Kersigo, J., Gray, B., Fritzsch, B. Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology. J. Vis. Exp. (49), e2451, doi:10.3791/2451 (2011).
Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.
1. Lipophilic Dye Injection
All tissue dissections and manipulations are carried out in animals fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) by transcardial perfusion using peristaltic pumps with appropriately sized needles. Tissue can be stored in 4% PFA in the refrigerator for up to 6 months. All preparations and manipulations are conducted in 0.4 % or higher PFA until mounting with glycerol for imaging. This amount of PFA effectively eliminates RNases and preserves the RNA for in situ hybridization.
All dyes should be stored in a dark closed compartment to minimize air circulation and exposure to bright daylight, as they are photosensitive. To avoid cross contamination, a separate set of instruments should be used to handle each dye.
Figure 1. Schematic overview of experiment. The ear is exposed to allow for visualization of all structures. Lipophilic dye is then injected and allowed to diffuse. After diffusion distinct neuronal populations can be seen labeled by the different dyes. Next, in situ hybridization (Sox2) is performed on the ear and labels mRNA expression. Immunohistochemistry is then carried out (Myo7, Tubulin) to visualize protein expression. Followed by histological sections in which both in situ hybridization and immunohistochemistry can be visualized.
2. in Situ Hybridization
In case you want to start with in situ hybridization (after which tracing with lipophilic dyes will be impossible), refer to tissue preparation in Part 1. Each wash, unless indicated, is carried out with 2 mL of solution at room temperature on an inverter/mixer. Be sure to work in a clean, RNase free environment.
|Table 1. Proteinase K digestion time on mouse tissue.|
|AGE (embryonic day)||TIME (minutes)|
*Samples can be imaged at this stage and/or after the immunohistochemistry step is added (Part 3 below).
Steps 1 and 2 can be omitted if Part 2 was completed. In that case, make sure that all glycerol or other mounting medium is washed off. Note that not all antibodies will still be able to detect their epitope after proteinase K digestion. We have a short list of antibodies that can be used in combination with in situ hybridization in mammalian tissue as they retain their specificity (see Table 2).
|Table 2. Antibodies that work with proteinase K digested tissue.|
|25-6790||Anti-Myosin-VIIA Polyclonal||Proteus Biosciences|
|25-6791||Anti-Myosin-VI Polyclonal||Proteus Biosciences|
|9661||Cleaved Caspase-3 Polyclonal||Cell Signaling|
|T7451||Anti-Acetylated Tubulin Monoclonal||Sigma|
Histology can be introduced after Part 1 to increase resolution of the lipophilic dye tracing in thick whole mounts such as juvenile brains or after completion of Parts 2 or 3. For serial brain sections we recommend the Compresstome VF-700 microtome (Precisionary Instruments Inc., Greenville, North Carolina) to obtain uniform section thickness. Frozen sections are less optimal due to greater dye leakage during the sectioning process 3. Preparation of serial sections and mounting in glycerol 4 is the preferred method of tissue preparation when whole mounts or surface mounts do not allow adequate visualization of tissue regions of interest. Tissue can also subsequently be embedded in epoxy resin and cut at 1-2 μm thickness using glass or diamond knifes for TEM. When using this technique most immunofluorescence will remain and is even more stable after plastic embedding, however, all lipophilic tracing will be entirely lost at this stage as they dissolve in alcohol and epoxy resin. Take precaution as samples will be light sensitive until they are embedded.
For Compresstome VF-700 microtome sectioning, follow the recommendations of Precisionary Instruments Inc.
5. Representative Results
Figure 2. Lipophilic dye placement and imaging in a mouse embryo. Three different wavelength lipophilic dyes were injected and incubated to allow the visualization of the trigeminal nerve (TN), glossopharyngeal nerve (GN), and vagus nerve (VN) central projections. A) Lipophilic dye placement into the peripheral portions of three cranial nerves to allow diffusion to the brainstem for visualization of their central processes. The TN is labled with NeuroVue Red, GN: NeurVue Maroon, and VN: NeuroVue Jade. B) Same mouse as in (A) after incubation. Some diffusion can be seen with brightfield microscopy. C) Same mouse as in (A and B). The hindbrain has been dissected and flat mounted. Some labeling of the central processes of the three nerves labeled can be seen with brightfield microscopy. D) Confocal image of (C). With use of the confocal specific neurons can be seen, and use of three different dyes allows for the assessment of the distribution of each population in relation to the others. Dyes were imaged sequentially. NeuroVue Jade was imaged at an excitation of 488 nm and emission 500-550 nm. NeuroVue Red was imaged at an excitation of 535 nm and emission 550-600 nm. NeuroVue Maroon was imaged at an excitation of 643 nm and emission at 650-700 nm.
Figure 3. Schematic overview of experiment. The ear is exposed to allow for visualization of all structures. Lipophilic dye is then injected and allowed to diffuse. After diffusion distinct neuronal populations can be seen labled by the different dyes. Next, in situ hybridization (sox2) is performed on the ear and labels mRNA expression. Immunohistochemistry is then carried out (Myo7, Tubulin) to visualize protein expression. Followed by histological sections in which both in situ hybridization and immunohistochemistry can be visualized.
In this video, we demonstrate a method to combine four techniques in order to maximize data collection and cohesiveness by correlating data within a given animal (figure 3). This approach will reduce the amount of breeding and thus time needed to obtain publishable data while improving information about co-localization and correlative effects of mutants. Although embryonic mice were utilized in this video, adult mouse as well as other animals such as chicken, xenopus, and zebrafish can be analyzed using these techniques as well. When using other species proteinase k digestion may need to be altered. Here we use different fluorescing NeuroVue dyes to specifically label up to three neuronal populations of interest. The benefit of these dyes is that they can be used in mutant mice, which may be problematic when relying solely on immunohistochemistry that may or may not be affected by the mutations to be analyzed, and they label neurons retrogradely and anterogradely 5. A recent study has also increased the number of neuron populations that can be labeled simultaneously to six 2. After, the lipophilic dye tracing the same tissue of interest can then be analyzed using in situ hybridization, for analysis of gene transcription and can be subsequently analyzed using immunohistochemistry for protein distribution. The latter analysis can be supplemented by detailed histology which can add to the phenotype characterization 6. This multifactorial analysis has the potential to gain new insights through correlative analysis within the same animal that may otherwise be improbable or at least in need of more extensive protocols and mouse breeding.
Mouse tissue was collected in accordance with the guidelines and regulations set forth by the University of Iowa Institutional Animal Care and Use Committee (ACURF 0804066).
Confocal images were obtained at the University of Iowa Carver Center for Imaging. Funding was provided by an SBIR grant (MH079805) to B.F. Additional support for breeding the mice for this project was provided by NIDCD (5R01DC00559007) to B.F.
|in situ Hybridization:|
|PC DIG wash + block buffer set||Roche Group||1585762|
|Premix 20x SSC Buffer||Roche Group||1666681|
|Proteinase K 20mg/ml||Ambion||AM2546|
|Diethyl Pyrocorbonate (DEPC)||Research Products International Corp.||D43060|
|Dextran Sulfate||Research Products International Corp.||D20020|
|BM Purple||Roche Group||11442074001|
|Anti-Dig-AP Fab fragments||Roche Group||11093274910|
|RNase A enzyme 10mg/ml||Fermentas||EN0531|
|RNase Away||Research Products International Corp.||7003|
|Salmon Sperm DNA 10mg/ml||Invitrogen||15632-011|
|Stericup Filter Unit 0.22Ám; 500ml||EMD Millipore||SCGVU05RE|
|Filter Discs 0.2Ám; 25mm||Whatman, GE Healthcare||6780-2502|
|Phosphate buffered saline PH 7.4 packets||Sigma-Aldrich||P-5368|
|Hoechst Dye||Polysciences, Inc.||9460|
|Poly/Bed 812 Embedding Media/DMP-30 Kit||Polysciences, Inc.||08792-1|
|Gluteraldehyde Solution 50%||Fisher Scientific||G151|
|Heat Block||Fisher Scientific||11-715-125D|
|Rotator||Thermo Fisher Scientific, Inc.||400110Q|
|Incubator set to 60°||Fisher Scientific||11-690-625D|
|NeuroVue dye: Maroon, Red, Jade||MTTI||FS-100(1,2,6)|
|1X phosphate buffered saline (PBS)||Sigma-Aldrich||P-5368|
|Dissecting scope||Leica Microsystems||M205 FA|
|Incubator set to 36°||Fisher Scientific||11-690-506DQ|
|Confocal Microscope||Leica Microsystems||LSM 510|
|Paraformaldehyde (4% and 0.4%)||Fisher Scientific||T353-500|