The purpose of in situ hybridization (ISH) is to visualize TKmRNA molecules within cellular structures. High temperatures are used in the ISH procedure, which places special demands on the immobilization of the cells. If the in situ hybridization method does not provide a positive hybridization signal, other techniques can be applied, such as mRNA amplification to detect the presence of low abundance of mRNA in the microscopic regions of neuronal protrusions (Miyashiroetal.1994).
Modern Neuroscience Research Techniques
Author(s): U. Windhorst & H. Johansson Translated by Z. Q. Zhao Jun Chen
Operation method
Experiments on in situ hybridization techniques for cultured neurons
Principle
flow chart
Materials and Instruments
Solutions and Buffers In Situ Hybridization Move I. Preparation of digoxin-labeled probes The cDNAs are cloned into the pBluescript vector. For in vitro transcription, the plasmid is linearized or PCR'd and the antisense RNA probe is synthesized by applying T3 or T7 RNA polymerase depending on the orientation of the inserted cDNA. After in vitro transcription, the DNA template is degraded with DNAase I (without RNAase) and the cDNA is hydrolyzed into a fragment of approximately 200 nucleotides. Linearization of the plasmid: The plasmid is cleaved with a suitable restriction endonuclease at the site immediately downstream of the stop codon of the insertion sequence. The DNA is then purified by phenol extraction and ethanol precipitation. PCR amplification of the insert sequence: Primers were designed according to the flanking sequences of the T3 and T7 RNA polymerase promoters in pBluescript, and the cDNA was amplified by PCR, and then the DNA was purified by gel electrophoresis. (A detailed description of these techniques can be found in Maniatisetal.1982). 1. The following substances are added to a sterilized microcentrifuge tube: 2. After brief centrifugation, incubate for 2 h at 37°C. 3. Add 1ul of DNAase I (without RNAase) and incubate at 37℃ for 10 min. 4. Probe hydrolysis: add the following substances to the tube: 5. Mix and ice bath, 30 min for probes less than Ikb, 60 min for probes greater than Ikb. 6. Warm to room temperature (room temperature is 25℃), add 20 mllmol/L MES. 7. Add 28 ml of 7.5 mol/L ammonium acetate and 412ul of ice pre-cooled ethanol to precipitate the probe (at least 30 min at -70°C and 2 h at -20°C). 8. Centrifuge for 10 min at 12OOOr/min in a bench-top centrifuge. 9. Discard the supernatant (be careful not to pour out the precipitate during handling) and wash the precipitate with 70% ice pre-cooled ethanol. 10. Centrifuge at 12000 r/min for lOmin, pour off as much ethanol as possible and withhold the precipitate. 11. The final product is 2~4RNA, dissolved in 25ul of DEPC-treated water. The mass concentration of the product is 80~160ng/ul. See Chapter 2 for quality control of the probe. DNA is denatured in 0.1~0.2md/L Na+ solution at 90~100°C, with the highest rate of reversion occurring at 25°C below the temperature of strand release (Tm). For in situ hybridization (ISH), this means that the hybridization time of trace samples in 65~75°C must be prolonged, however such high temperatures can seriously affect the morphological structure of cells. The current solution to this problem is mainly the application of organic solvents that reduce the thermal stability of nucleic acid double strands, so that in situ hybridization can be performed at lower temperatures. Formamide has been widely used because at a certain concentration it linearly decreases the Tm of the DNA double strand, i.e., for every one percentage point increase in the concentration of formamide, the Tm decreases by 0.72°C. This is because formamide can be used as a solvent to reduce the thermal stability of the DNA double strand at a certain concentration. The effect of formamide on Tm can be calculated using the following equation. RNA can form stable double strands with RNA, therefore, when we use RNA probes for in situ hybridization, we need strict hybridization conditions. In our experience, hybridization mixtures containing 60% formamide at 50°C give good results. Add to a 50 ml sterilized polypropylene tube: Hybridization buffer should be protected from light and stored in a refrigerator. Acid-base cleaved salmon sperm DNA is prepared as follows: 1. Add Ig salmon sperm DNA to a 50 ml sterilized tube, add 15 ml of DEPC-treated water, and soak for 15 min to 2 h. 2. Add 2.5 ml of 2mol/L hydrochloric acid and leave it at room temperature. 2.5 ml of Ig salmon sperm DNA should be added to the sterilized tube. 2.5 ml of 2mol/L hydrochloric acid should be added and left at room temperature. 2.5 ml of 2.5 ml of 2mol/L hydrochloric acid should be added to the sterilized tube. 3. Add 5.0 ml of 2.0 mol/L NaOH, shake the DNA to re-dissolve it, and incubate at 50℃ for 15 min to accelerate the dissolution of DNA. 4. DEPC treated water was used to dilute the mixture to 175 ml to ensure that no particles were present. 5. Add 20 ml of Imol/L TrisHCl (pH 7.4). 6. Adjust the pH of the DNA solution to 7.0~7.5 with 2mol/L hydrochloric acid. Filter the solution through a sterilized microporous filter to remove any clumps of precipitate: measure the absorbance of the solution at 260 nm. Pipette 20ul of DNA solution into 980ul of water. Pipette 20ul of DNA solution into 980ul of water. 40 times the absorbance is the mass concentration of DNA in 1ug/ug. Dispense into a solution with a mass concentration of 4 mg/ml and store at -20 °C. For isolation and culture of neurons see the methods provided in Chapter Ten. Because we chose to use RNA probes for in situ hybridization, measures must be taken to prevent degradation of the RNA. Therefore, it is important to wear gloves, use sterile pipette tips, microcentrifuge tubes, and RNAase-free buffer [0.25% DEPC in redistilled (distilled) water, incubate overnight at 37°C, and then autoclave]. All solutions should be filtered through a 0.22um pore size filter, as any small particles in the petri dish can damage neurons. 1. Cells should be fixed for at least 2 h. Fixative should be added slowly to the culture dish to avoid stripping the neuronal cells when changing the culture medium. For fixation of invertebrate (e.g. mollusc) neurons, a mixture of 1% paraformaldehyde and 1% acetic acid is used. While for vertebrate neurons, 4% paraformaldehyde is recommended and the concentration of acetic acid can be increased to 5% (Dirks 1996). Acetic acid is a good fixative for nucleic acids. 2. A final concentration of 0.5% NonadetNP40 is added to the fixative and incubated for 30 min. 3. Buffer 1 is washed twice for 10 min each time. 4.(Optional) Treat the cells with 0.1%~0.005% pepsin solution (dissolved in 0.2mol/L hydrochloric acid) for 10 min at 37°C. This step allows the probe to easily access the target mRNAs in the cells. In our experiments, we omitted the proteolytic hydrolase treatment step altogether, and we did not find any significant loss of signal. Thus, we omitted steps 5 to 8. 5.2% polymerase fixation for 4 min. 6.1% hydroxyammonium chloride (dissolved in buffer 1) for 15 min. 7. Wash the paint with Buffer 1 for 5 min. 8. Pre-hybridize in probe-free hybridization buffer and incubate at 50°C for lh. This step may not be necessary, but the need for pre-hybridization should be checked each time depending on the type of cells selected. 9. Hybridization with hybridization buffer. Typically IOOul of Hybridization Buffer contains 1ul of probe. Prior to hybridization, heat the Hybridization Buffer to 95°C and then immediately place on ice to cool rapidly. Incubate at 50°C for at least 3 h, or overnight. Place the petri dishes in an airtight container with wet filter paper to prevent evaporation of the Hybridization Buffer. Add enough Hybridization Buffer to ensure that the cells are completely covered, we usually add 100ul of buffer to each well. 10.2XSSC Wash the cells briefly 2 times for no more than 2 min each time. 11. Strict washing. Wash the cells 3 times for 20 min at 50°C with a mixture of 2XSSC and 50% methylglutamine to remove unhybridized probes from the cells. If the background is still high, treat with RNAase A (see step 12), which removes all unhybridized probes. 12. (Optional) Add RNAase A (100 ng/ul in Buffer 1) to 2XSSC and treat the cells for 25 min. followed by a rigorous wash (see step 11). In other words, if RNAase is used to treat the cells, the cells need to be washed rigorously twice before and twice after treatment with a mixture of 50% formamide and 2XSSC. 13. 2XSSC washes the paint for 5 min. 14. Buffer 1 Paint Wash, 2X5 min. 15. Wash with TBSGT buffer for 15 min. 16.Add alkaline phosphatase cross-linked anti-digoxin antibody (diluted 1:500 with TBSGT buffer) and incubate at room temperature for 2 h or overnight at 4℃. 17. Wash twice with buffer 1 for 5 min each time. 18. Buffer 2 2 washes, 5 min each. 19. Incubate in alkaline peptidase substrate: add 4.5ul of NBT and 3.5ul of BCIP to 1 ml of Buffer 2. If the neurons have endogenous alkaline phosphatase activity, add 10 lmol/L levamisole. Add approximately 0.5 ml of substrate to each dish. The incubation time should be adjusted according to the amount of transcripts in the cells, from a minimum of 15 min to overnight, and should be protected from light. The alkaline phosphatase substrate used is catalyzed to produce a dark purple reaction product. If a fluorescent substrate is required, solid red TR (1.0 mg/ml in buffer 2) or naphthol (0.4 mg/ml in buffer 2) can be used, which can be visualized as a burst of light through the rhodamine filter of a fluorescence microscope. 20. After the results were observed with the naked eye, the reaction was terminated by treatment with Tris-EDTA buffer for 15 min. 21. Rinse the cells with redistilled (distilled) water. 22. Seal with sealer aquamount or glycerol. 23. Observe the cells under a microscope We used in situ hybridization to study the distribution of mRNA encoding neuropeptides in neuronal protrusions of neurons from vertebrate solid snails in primary culture. The question of interest to us was whether the transcript was evenly distributed throughout the neural protuberance or clustered in a specific tiny region. In situ hybridization experiments showed that the mRNA was particularly abundant at the growth cone and varicosities (Fig. 3-1) Common Problems Note: All materials should not contain RNAase, and manufacturers' sterilized plastics should be used whenever possible. For more product details, please visit Aladdin Scientific website.
RNA polymerase Digoxin-UTP labeling mixture
