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In Situ Hybridization, In Situ Transcription, And In Situ Polymerase Chain Reaction, L. E. De Bault, J. Gu

Scanning Microscopy

In situ hybridization, in situ transcription, and in situ polymerase chain reaction (PCR) are techniques used to detect DNA and RNA sequences within a cell or tissue structure. These three in situ methodologies employ the principles of recombinant DNA to form double-stranded hybrids of DNA-DNA, DNA-RNA, or RNA-RNA. The essence of in situ hybridization (ISH) is the hybridization of a labeled probe to a complementary target sequence, whereas in situ transcription (IST) is the synthesis of complementary DNA incorporating a label directly on the target DNA or RNA within a cell or tissue. In the case of in situ PCR …

Preparation Of Samples For Polymerase Chain Reaction In Situ, Gerard J. Nuovo

Scanning Microscopy

The purpose of this paper is to describe the key variables in sample and reagent preparation needed for successful polymerase chain reaction (PCR) in situ. Tissue or cell preparations should be fixed in a cross linking fixative, such as 10% buffered formalin, preferably from 15 to 48 hours. Tissues should be embedded in paraffin; cell preparations can be fixed when near confluence, then physically removed and processed. When possible three samples (4 μM tissue sections or 1-5000 cells) should be placed on silane coated glass slides. Digestion in pepsin (2 mg/ml) for 30 min is adequate for DNA detection …

Simultaneous Identification Of A Specific Gene Protein Product And Transcript Using Combined Immunocytochemistry And In Situ Hybridization With Non-Radioactive Probes, Gwen V. Childs

Scanning Microscopy

Simultaneous identification of messenger RNA (mRNA) and proteins in the same cells or tissues is a valuable tool to help the cell biologist evaluate the cell secretory cycle. Some cells may produce the mRNA and delay the production of the proteins. Alternatively, the proteins may be rapidly secreted. Other cells may produce both in sequence within the same time frame. Because of this difference, some cells can only be identified by their mRNA product. Others may have both products. This presentation describes a non-radioactive approach to the detection of both products with dual-peroxidase labeling protocols in use in this laboratory …

Preparation Of Plasmid Dna In Transfection Complexes For Fluorescence And Electron Spectroscopic Imaging, Marek Malecki

Scanning Microscopy

The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy.

Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first method tetramethylrhodamine, nanogold, and ferritin were linked through streptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent derivatives was studied in living cells with fluorescence microscopy. Then, selected cells were rapidly cryo-immobilized. …