In vivo immunostaining can be used for the detection of modified poxvirus Ankara (MVA) and also for the identification of proteins expressed on the cell surface or in the cytoplasm. It is of course also applicable to standard poxvirus strains. Source: Compact Laboratory Guide to Molecular Biology (Fifth Edition)
Operation method
basic program
Principle
In vivo immunostaining can be used to detect modified poxvirus Ankara (MVA) because MVA is unable to form separate, readily recognizable phage spots and this technique does not require the utilization of screened marker genes.
Materials and Instruments
Confluent Piece-Grown CEF Cells Move 1. Trypsin digestion of confluent growing CEF cells was performed, and they were cultured in cutin A-coated 6-tissue culture plates until the cells grew to confluence. 2. Remove the transfected cell lysate on ice and sonicate on a cup sonicator at maximum power for 30 s. Add 2 ml of complete MEM-2.5 medium containing 100, 10, 1, 0.1 μl of cell lysate to each well, repeat for two wells for each concentration, shake gently to mix, and incubate for two days. 3. Immunostain the unfixed cells with primary antibody against the exogenous gene expression protein. 4. Aspirate the medium, observe the cells under an inverted microscope, pick the immunostained spots with a sterile toothpick, break the toothpick, and put the sterile part of the toothpick into vials containing 0.5 ml of complete MEM-2.5 medium. 5. Repeatedly lyse the cell suspension by freeze-thawing: freeze the cells with dry ice/ethanol, then thaw the cells with a 37°C water bath and vortexing. This is done 3 times. The cells are sonicated on a cup sonicator at maximum power for 30 s. The cells are then infected with new cells. 6. Infect new CEF cells with the recombinant MVA virus and perform a second round of phagocytosis and three rounds of phagocytosis according to steps 2-5. 7. 7. Infect CEF or BHK-21 cells cultured in one well of a 6-well tissue culture plate coated with cutin A with 0.25 ml of plaque-purified recombinant MVA virus and add MEM-2.5 medium to 2 ml. Incubate for 2 days. 8. Aspirate 1 ml of plaque-coated recombinant MVA virus into a 6-well tissue culture plate. 8. Aspirate 1 ml of cell-covered medium and use a cell spatula (or the plunger of a 1 ml syringe) to pass the cells into the remaining 1 ml of medium and transfer to a vial. Repeatedly freeze-thaw the cells as in step 5 to obtain a cell lysate. 9. 0.5 ml of cell lysate is inoculated onto CEF and BHK-21 cells cultured in 75 cm2 flasks containing 15 ml of MEM-2.5 medium and the virus is amplified. 2 days later, the cells are harvested and lysed according to Step 8. 10. 0.5 ml of cell lysate from step 9 is used to inoculate CEF and BHK-21 cells cultured in 150 cm2 flasks with 30 ml of MEM-2.5 medium. 11. Virus titers are measured and the virus is detected. 11. Determine the viral titer and prepare a large reservoir of recombinant viral MVA, divide into small portions, and store at -70 °C. Caveat 1. if the target protein is expressed in the cells, aspirate the solution from the wells and place the plates at -70°C for 1 h. Thaw and stain, a step that ruptures the cells in situ to allow antibody penetration. 2. In this experimental protocol, phagolysin purification was performed with liquid instead of agarose as a cover layer to facilitate immunostaining. To test for stability and purity, another culture plate was used for immunostaining. Spots that were not stained resulted from cytopathic lesions due to wild virus or unstable recombination. For more product details, please visit Aladdin Scientific website.
Fully MEM-2, MEM-2.5 and MEM-10 media transfected cell lysates Dry ice/ethanol Anti-exogenous gene expression protein primary antibody Horseradish peroxidase-linked secondary antibody
Knife bean protein A-coated 6-well tissue culture plates Cup sonicator Inverted microscope Sterile toothpick Cell scraper/piston of 1 ml syringe 75 cm2, 150 cm2 Tissue culture flasks