What is disadvantges of fluorescence microscopy?

Answer 1

Florence microscopy has 3 main different way to make the cell fluorescence each with the disadvantages.

Label protein outside the cell and microinject it - Takes a long time for little results

You can use Immunofloresence which is faster by direct or indirect. Indirect you get better results through more floresence and takes less time because you can order the antibodies off the internet.

You can use GFP gene and insert it into the cell. What you can do is have a plasmid transfect it then the protein will glow - This has a downside if the protein doesnt fold.

If you are doing the immunofloresence you need to prepare the cell. You need to kill it to use the antibodies. You use formaldhyde or gluteraldehyde or methanol for this. This will but everything in place (cytoplasm wont move) then use a detergent to punch holes in the membrane. Then you use your anti-bodies to go into the cell and attach to your primary proteins. that you want to see.

To make these antibodies you can proceed either by monoclonal or polyclonal. inject the rabit or w/e animal with an antigen. You will get antibodies forming with the epitop to that antigen that antigen should be the protein you want. Polyclonal have more then 1 epitope. and you need the rabit to do this.. if it dies bye bye antibodies disadvantage

Monoclonal have Hybridomas which are immortal cell lines, they regognize only 1 epitope.

Myeloma cell with a lymophocytes.

To make sure you only have there hybridoma cell you can put them in HAT which will kill the myloma cells and you only get hybridomas.