Modern Neuroscience Research Techniques
Author(s): U. Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun
Operation method
Intracellular injection of mRNA Move I. Preparation of capped mRNA for intracellular injection MATERIALS 1. Add the following substances sequentially in a final volume of 20 ul and operate on ice: 2. Incubate at 37℃ for lh. 3. Add Iul DNAase I (without RNAase) and incubate at 37℃ for 10 min. 4. Purify mRNA with RNaid kit. 5.25ul of sterilized water (without RNAase) to dissolve RNA. (For solutions not mentioned, see Method 1, "In situ hybridization of cultured neurons".) 1. Isolate the neuron from the CNS, keeping one long segment of its axon intact. 2. Incubate the neurons in conditioned medium for i8~48 h, until sufficient protrusions have grown from the original axon. 3. Using a sharp glass electrode, transverse cut the axon from the cytosol at a distance of about one cell diameter. 4. Fill the glass electrodes with mRNA (mass concentration of 200?50 ng/pd) using a microsampler. Fill only the electrode head with mRNA solution (approximately 0.3ul per electrode). 5. Puncture the axon and inject the mRNA into the axon with a 20 psi pulse of 1-15 S. The mRNA is then injected into the axon. White droplets of mRNA can be observed under a phase contrast microscope. 6. Warming. The incubation time depends on the cell type and the type of mRNA used. In our experiments, mRNAs encoding ovarian hormone precursors from the genus Spiroplasma were used, and their transformed proteins could be detected by incubating the cells for 2 h after injection. 7. Fixed and permeabilized cells were processed according to steps 1-3 of the Cultured Neurons:ISH method. 8. Wash with TBSGT twice for 5 min each time. 9.incubate with primary antibody for 2 h at room temperature or overnight at 4℃. 10. Wash with TBSGT twice for 10 min each time. 11. incubate with secondary antibody at room temperature for 1~2 h. In our experiments, we used FITC crosslinked secondary antibody or enzyme crosslinked antibody. The procedure for cross-linking the secondary antibody with alkaline phosphatase can be found in Method i "In situ hybridization of cultured neurons". 12.TBSGT buffer wash丄 times, 5 min. 13. Wash with water 2 times, each time I0 min. 14.75% glycerol (dissolved in water) sealing, glycerol solution contains 0.5% ethylenediamine to slow down the discoloration of FITC. 15. Inverted fluorescence microscope observation. To investigate whether mRNA in isolated axons can be converted into protein, we injected mRNA encoding the precursor of ovotropin (ELH) into axons of PedalA cells. We first confirmed that these cells do not express the ELH gene. The translation of the injected mRNA was then detected by immunocytochemistry using antiserum to ELH. The results showed that isolated axons were able to translate the injected mRNA into proteins detectable by immunocytochemical methods. The translated products were mainly localized to growth cones and varicosities (Figure 3-7). For more product details, please visit Aladdin Scientific website.
