RESUMEN
Immunofluorescence staining is a very common method for the subcellular localization study of proteins. A tissue-chopping-based immunofluorescence staining method for chloroplast proteins overcomes the restriction of plant cell wall, makes the operation simpler, and uses less experimental materials. Here we provide some improvements for this method. We found that the stained tissues can be directly observed with a confocal microscope without tissue lysis. Samples maintained at a low temperature (0-4 °C) throughout the process can reduce the intensity of chlorophyll autofluorescence and the background signal. A low temperature is also good for the storage of the sample. Fluorescence signal of the stained samples can be kept for several weeks if they are stored at -20 °C. FtsZ is an essential component of the chloroplast division apparatus. We demonstrated this method with the immunofluorescence staining of FtsZ1 in wildtype Arabidopsis and some chloroplast division mutants. We also successfully tested this method by the immunofluorescence staining of FtsZ1 in many other plants, including woody plants. With these procedures, the performance of tissue-chopping-based immunofluorescence staining method are further improved.
RESUMEN
Immunofluorescence staining is an important method for detecting the localization of proteins in the cell. It is also frequently used in the localization study of chloroplast-division proteins. Although this method has been improved before by using protoplasts, it still has some limitations. Now we developed a new method to make it much easier. We just broke the plant leaf tissue with a serrated blade, stained the samples directly, and simply lysed the tissue into separatable cells. The localization of the target protein can then be observed with a clear view. Since this method directly uses broken leaf pieces, it is very fast. It can also be applied to the plants in which protoplasts are difficult to prepare. We first used this method to observe the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis. A ring was clearly seen in the middle of chloroplasts. In addition, we used this method to analyze the localization of FtsZ1 in arc3 and pdv2 mutants, as well as in dozens of other species, including some woody plants. This new immunofluorescence staining method is not only easy to use, but also has a wide applicability in various plants.