Electron microscope in situ hybridization technique experiment
Electron microscope in situ hybridization technique experiment
Electron microscopy in situ hybridization experiments can be used to (1) study the subcellular localization of specific nucleotide sequences; (2) many researchers have expanded from optical microscopy to the observation of ultrastructures. (3) In particular, the use of digoxin or biotin as a non-radioactive probe for reporter molecules does not have to require prolonged exposure, as is the case with radioactive probes, and thus the whole process can be completed within 24 h.
Operation method
Electron microscope in situ hybridization technique experiment
Principle
Theoretically, the pre-embedding in situ hybridization technique is more sensitive than the post-embedding in situ hybridization technique because the former allows the observation of signals from the entire thickness of the section. However, the probe does not always penetrate the entire section thickness, especially with longer probes. To increase penetration, freeze-thawing or protease and detergent treatments are often applied, which can lead to the loss of some intracellular components, such as ribosomes, and in severe cases to morphological changes in the ultrastructure. An even more unfavorable factor is the inability to display hybridization probes with larger gold crosslinks (>5 nm) without robust permeabilization treatments. As an alternative, peroxidase crosslinks and ultramicro gold crosslinks (<lnm) can be used. Peroxidase can be colored with diaminobenzidine, but has the disadvantage that the resolution is too low and the reaction products cannot be quantified. Ultramicrocrosslinks, on the other hand, are too small to be observed directly under the microscope and must be enhanced by silver staining to be visible (Danscher 1981), but this process may produce silver particles of irregular sizes, which can hinder the accurate localization of the target. Moreover, silver staining enhancement has been reported to negatively affect the ultrastructure (Eggeretal.1994;Macvilleetal.1995). Therefore, the post-embedding hybridization technique is comparatively a better method, which maintains a good ultramicro-morphological structure with reasonable sensitivity. Since only the surface of the section is labeled, larger gold crosslinks can also be used to display the hybridization probe. Hydrophilic resins (e.g., LowicrylK4M, HM20, LRWhite, LRGold, and Bioacryl) are clearly superior to traditional EM embedding agents (e.g., Epon and Araldite) because the hydrophilicity of the former greatly enhances the ability of the probes to approach the target nucleic acid sequence. Some non-embedding methods, such as the often-mentioned frozen section technique, combine the advantages of both pre-embedding and post-embedding methods, i.e., high probe proximity to target nucleic acid sequences and good retention of ultrastructure. However, this method also has disadvantages, such as the expensive equipment required, the need for skilled technicians to operate it, and the fact that the sections are very sensitive to denaturation treatments to the extent that some of the ultrastructure and target nucleic acid sequences can be lost. The choice of the above three methods depends on the needs of the researcher and the sample and should be treated separately on a case-by-case basis. The advantages and disadvantages of these three methods have been summarized in Table 3-1. In our experience, the non-embedding method gives the best results in terms of sensitivity and maintenance of ultrastructure. Moreover, it can be easily combined with immunocytochemistry to identify other substances (e.g., neuropeptides, etc.) in the same sections. We have used this technique to study the ultrastructural localization of mRNA encoding neuropeptides in axonal sections of molluscan neurons.
Materials and Instruments
Tissue Samples Move I. Tissue fixation of ultrathin frozen sections 1. Use clean glassware or disposable utensils to prepare fixatives 2. Use freshly prepared fixative. 3.2% to 4% paraformaldehyde and 0.2% glutaraldehyde to fix the tissue. Glutaraldehyde is necessary to ensure good ultrastructure. However, due to the cross-linking properties of the fixative, higher concentrations (>0.5%) severely impede penetration of the probe. Therefore, for each sample to be used in the study, a balance must be found between ultrastructural preservation and probe penetration. Small pieces of tissue are fixed by immersion. For large tissues, such as rat brain, perfusion fixation is recommended. 1. Fix small pieces of tissue by immersion at 4°C overnight. 2. Wash the tissues twice with Imol/L phosphate buffer for 10 min each time. 3. Wash the tissue twice with 0.1 mol/L phosphate buffer containing 0.15% glycine for 10 min each time. soak the tissue in 2.3 mol/L sucrose PBS for at least 4 h, rolling the tubes continuously during the soak. Tissue blocks were fixed to the specimen support before sectioning; we generally used copper nails for fixation. 1. Roughen the surface of the nail with sandpaper. 2. Wash the nails to remove small metal fragments. 3. Wash nails with 70% ethanol and dry with absorbent paper. 4. Remove the tissue from the sucrose solution and position the tissue on the nail using a dissecting microscope.... Absorb excess sucrose with blotting paper. 5. Using clean forceps, place the nail with the sample into liquid nitrogen. 6. Store the sample in liquid nitrogen until use. 1. When transferring tissue samples from the liquid nitrogen tank to the ultrathin slicer, make sure there is no melting. 2. The ideal temperature for ultra-thin frozen sectioning is 100~120℃. 3. Finish slicing with dry knife. 4. A thick section (500 nm) is often made to localize the tissue and to check fixation. These thick sections can be stained with 1% toluidine blue solution. 5. Fine trimming of specimen edges improves the quality of ultrathin sections. This can be accomplished with a razor blade or glass cutter. 6. For thin sections (40~80nm), the use of a diamond knife is recommended. 7. Use an inoculation ring to separate the sections from the blade. The inoculation ring is an I5 cm plastic handle attached to a small metal collar (2 mm diameter) filled with 2.3 md/L sucrose or sucrose/methylcellulose mixture. 8. After collecting the sections with the inoculation ring, allow them to thaw briefly and transfer them immediately to a sieve. 9. Collecting sections with the sucrose/methylcellulose mixture has the two advantages of better and more intact retention of morphological structure, and the sieve mesh can be stored in the refrigerator for at least 3 months. 10. Use clear plastic-coated or carbon-coated nickel screens; copper screens oxidize during processing and form dirty precipitates that can interfere with electron microscopy imaging. Place the sieve (slice side down) on a covered petri dish containing 2% gelatin solution, place on a hot plate (37°C) for 5 min, and then incubate at 37°C for 20 min. The following steps were done in droplet liquid. Spot small drops of reagent (5-10ul for probe and antibody solution, 100ul for washing) onto the sealing film wax and place the screen section face down on the small drop. Protein A-gold cross-links were chosen for this method. Because protein A has a high affinity for rabbit IgG and a low affinity for goat IgG, and anti-digoxin antibodies are mostly goat antibodies, a rabbit-anti-goat secondary antibody was used as an intermediary for signal amplification. 1. incubate in 0.15% glycine PBS for 5 min twice. 2. Incubate with 2XSSC for 5 min twice. 3. Pre-hybridize in probe-free hybridization buffer for 20 min. 4. Add O.Ig probe solution to 10ul hybridization mix. 5. Place the hybridization screen in an airtight container with a wet filter paper to prevent evaporation of the hybridization buffer and leave at 50C for 3 hrs. 6. Incubate the Imin with 2XSSC. 7. Wash twice with a mixture of 50% formamide and 2XSSC for 5 min each time. 8. In a closed and humidified container, wash rigorously with a mixture of 50% formamide/2XSSC 3 times at 50°C for 20 min each time. 9. Wash twice with 2XSSC for 5 min each time. 10. 3 washes with PBS containing I% BSA for 5 min each. 11. Wash with PBS containing 1% fish gelatin for lOmin. 12. Incubate in goat anti-digoxin antibody (1:100 dilution in PBS containing 1% fish gelatin) at room temperature for Ih or overnight at 4C. 13. Wash with PBS containing 1% fish gelatin for 5 min. 14. Wash 3 times with PBS containing 1% BSA for 10 min each time. 15. Wash with PBS containing 1% fish gelatin for 5 min. 16. Incubate in rabbit anti-sheep antibody (1:100 dilution in PBS containing 1% fish gelatin) for 30 min. 17. Wash with PBS containing 1% fish gelatin for 5 min. 18. Wash 3 times with PBS containing 1% BSA for 10 min each time. 19. Incubate in protein A-colloidal gold crosslinker (diluted in 1%BSA PBS, according to the instructions for use) for 20 min. 20. Wash 3 times with PBS for 15 min each time. 21. Fixed in PBS containing 1% glutaraldehyde for 10 min. 22. Wash with PBS for 5 min. 23. Wash with distilled water 5 times, each time with Imin. 24. Incubate in 2% UO2 acetate for 5 min. 25. Wash 2 times with Uranyl acetate/methyl cellulose, each time Is. 26. Embed the sections in uranyl acetate/methylcellulose and place on ice for 10 min. 27. Remove the screen from the liquid droplets of methylcellulose with an inoculating loop. 28. Remove excess methylcellulose with filter paper, leave the sieve on the inoculation ring and let it air dry for 30 min. 29. Transfer the sieve to the sieve box. Electron microscopic in situ hybridization (EM-ISH) is conveniently combined with immunocytochemistry by following step 22 above: 1. 3 washes with PBS containing 0.15% glycine for less than 10 min each. 2. Wash with PBS containing 1% fish gelatin and I% BSA for 5 min. 3. Incubate with antibody (rabbit antibody, diluted in 1% fish gelatin and 1%BSA in PBS) for 45 min. 4. Wash 5 times with PBS containing 1% BSA, each time with Imin. 5. Incubate for 20 min with Protein A-Colloidal Gold Crosslinks (diluted in 1%BSA in PBS). Different sizes of gold can be used to compare the display of ISH signal. 6. Proceed to step 20 of the electron microscopy in situ hybridization method. Previous studies have shown that in the molluscan vertebrate solid snail, the axons of neurons capable of synthesizing orexigenic hormone (ELH) contain a large number of mRNAs encoding ELHVanMinnen1994). We used EM-ISH to study the ultrastructural localization of these mRNAs encoding neuropeptides. First, we investigated the localization of ELH transcripts within the cytosol of synthetic ELH neurons. As expected, they were found to be predominantly associated with the rough endoplasmic reticulum membrane (Figure 3-2A). Other organelles (e.g., mitochondria, Golgi, secretory vesicles) did not show the presence of ELH transcripts, as can be observed in Figure 3-4, where the above organelles did not show the co-localization of ISH signals (small gold granules) and ELH-immuno-responsive substances (large gold granules). We then examined the precise localization of ELH transcripts within neuronal axons and found that they were predominantly located in the axon plasma (Fig. 3-3), with no presence of their transcripts in ELH-containing nucleus accumbens vesicles, in accordance with previous reports (Dirks 1996). In our experiments, we mainly observed the ultrastructural localization of the encoded ELH mRNA and also found that goat anti-digoxin antiserum cross-reacts with ELH-containing nuclear dense vesicles. To avoid this problem, we used a mouse anti-digoxin monoclonal antibody (step 12), and then replaced the rabbit anti-goat antibody with a rabbit anti-mouse antibody at step 16. The above data further illustrate the need for proper control experiments when performing ISH. Caveat The temperature for hybridization and prehybridization can vary from 55 to 60 degrees, with lower temperatures resulting in better probe binding and higher temperatures resulting in less background.Source Modern Neuroscience Research Techniques Author:U.Windhorst&H. Johansson Translated by Zhao Zhiqi Chen Jun Common Problems This method has high sensitivity and specificity, and can further explore the functional expression of cells and their regulatory mechanisms from molecular water. It has become an important tool in today's cell biology and molecular biology research. For more product details, please visit Aladdin Scientific website.
Proportioning solutions
Frozen ultrathin sectioning machine Electron microscope Nickel mesh (75 mesh)
Source: Modern Neuroscience Research Techniques by U.Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun