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Fluorescence in situ hybridization [fish] in uncut plant cells using oligonucleotide DNA probes
| Content Provider | Semantic Scholar |
|---|---|
| Author | Smoliński, Dariusz Jan Wróbel, B. |
| Copyright Year | 2002 |
| Abstract | In plant cells, the analysis of gene expression at the transcription level using in situ hybridization is limited by the cell wall, which makes it impossible for the probes to penetrate the cell. The most frequently used solution for this problem is to embed and cut plant material. Such semi-thin or ultra-thin preparations can then be used for in situ hybrydization. In this study, we have attempted to overcome the cell wall barrier in order to spatially locate U2 snRNA in uncut meristematic lupine root cells using a confocal microscope. Root meristems were fixed in 4% paraformaldehyde (PBS buffer pH 7.2), subjected to enzymatic digestion (in a mixture of pectinase and cellulase in citrate buffer pH 4.8), and spread on microscope slides. The preparations were then washed with a Triton X100 solution and air-dried. In order to localize U2 snRNA, we used digoxygenin labeled oligonucleotide probes, chemically at the 5' and enzymatically at the 3' end (using the TdT enzyme). Then we used a primary mouse anti-DIG antibody and a secondary anti-mouse antibody labeled with Cy3 fluorochrome. We observed signals indicating the site of U2 snRNA location in the nuclei in the CB area and in specles and in lupin cell cytoplasm. This type of U2 snRNA location concurs with the observations of other researchers for plant and animal cells. The oligonucleotide probes introduced by us instead of the antisense RNA used so far in in situ hybridization on uncut plant cells eliminate the laborious procedure used for protecting cells from the degrading activity of RNAses, and at the same time yield very strong FISH signals. |
| File Format | PDF HTM / HTML |
| Volume Number | 07 |
| Alternate Webpage(s) | http://www.cmbl.org.pl/pdf/Vol7_suppl_p325.pdf |
| Alternate Webpage(s) | http://es.cmbl.org.pl/pdf/Vol7_suppl_p325.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
