Fixation situ hybridization

Bouin s solution is one of the traditional ways to harden cell pellet. Some cytologists believe it provides the best cellular details, especially nuclear features in cell blocks.28 The major steps are (1) After centrifugation, fix the cell pellet with Bouin s solution. (2) After 2h, discard the solution. (3) Remove the hardened cell pellet from the tube, wrap it with lens paper, and transfer it into a cassette for further processing. We have been using this method for many years. In our experience, most of the time, ICC results are consistent with IHC from the surgical specimen. The biggest drawback of this method is the toxicity of Bouin s fixative which creates biohazard and safety issues for the laboratory. We also found cell blocks gave poor fluorescence in situ hybridization (FISH) results after Bouin s fixation. 

Armed with the common techniques of molecular biology and immu-nocytochemistry, an investigator is in a good position to apply in situ hybridization to EM for localization of nucleic acids at the ultrastructural level. McFadden (9) has a review on such use of in situ hybridization techniques. In the review, McFadden has included details of some of his laboratory protocols needed in in situ hybridization from fixation and labeling to probe labeling to the hybridization steps for localization of specific RNAs at the EM level. His protocol for hybridization is outlined below ... 

Proper fixation is one of the most critical steps in an in situ RT-PCR or in situ hybridization experiment, because each tissue type must have optimized fixation conditions. Particularly archival tissues may require individual specific treatment in order to meet with in situ experimental requirements. Errors in fixation will only be discovered after the entire hybridization process has been completed (see Note 10). 

The fixation of CaSki cells in aldehyde has been shown to enhance the sensitivity of the detection of human papillomavirus type 16 by in situ hybridization compared with fixation by acid/alcohol (9). 

The role of high temperatures in the breakdown of protein crosslinks introduced by aldehydes has been discussed earlier in this book. The aforementioned combined technique is especially effective in increasing the in situ hybridization signal in archival tissues, the fixation history of which may or may not be known. This protocol also detects low levels of nucleic acids in the tissue, which may not be detectable with heating or enzyme digestion alone. 

The distribution of cannabinoid receptors has been extensively mapped by quantitative autoradiography, in situ hybridization, and immunocytochemistry. Each of these techniques has its strengths and weaknesses. Properly calibrated, quantitative autoradiography provides the best measure of absolute receptor density. Nonetheless, its spatial resolution is limited and specificity depends on the ligand used. In situ hybridization identifies the cells synthesizing CB i mRNA. However, mRNA levels and protein levels may not necessarily correlate. Immunocytochemistry provides outstanding spatial resolution however, fixation artifacts and unanticipated antibody crossreactivity must be assiduously avoided. For the most... 

The significant advantages of the described protocol rest in the versatility of probe design, the increased tissue permeability and hybridization efficiency because of the small probe size and, finally, the superior tissue preservation as a result of the rapid freezmg/dehydration and vapor-induced fixation. In addition, tissue preparation with the proposed protocol is compatible with immu-nocytochemistry. It is possible, therefore, to obtain serial consecutive paraffin sections from the same CNS and to combine in situ hybridization with immunocytochemistry for direct comparison of the signal from the two techniques in the same cells. For a more comprehensive and detailed review of in situ hybridization m general, the reader is referred to the excellent recent practical guide by Leitch et eil.(l) and also to the seminar series by Harris and Wilkinson (2). 

Sample Preparation and Fixation Methods for Whole-Mount in Situ Hybridization... 

Successful application of this experimental approach depends on several factors synthesis of high-quality hybridization probes, appropriate fixation of the sample, the hybridization procedure, and the fluorescence microscopy approach used to image the specimen. In adapting the technique of three-dimensional in situ hybridization to different organisms and tissue types, the simplest and most invariant aspect of the technology has proved to be the hybridization procedure. Probes must be developed on a custom basis to address the particular questions of the investigator, and equally crucially, fixation conditions need to be adapted with special attention to the physical attributes of the individual specimen. However, once appropriate preparation conditions are established for a particular type of sample, it has been unnecessary to reoptimize the basic hybridization protocol. We discuss each of these experimental issues separately below. 

Two protocols are provided for fixation of Drosophila embryos. Because these embryos are surrounded by both a chorion (eggshell) and an impermeable vitelline membrane, they require special steps to remove these barriers prior to immunostaining or in situ hybridization. Typically, the chorion is dissolved by treatment with sodium hypochlorite (commercial bleach) and the vitelline membrane is removed, either concomitant with or after fixation, by a combination of temperature and osmotic shock. The heptane in these procedures permits the fixative to pass through the vitelline membrane. 

This is a general protocol for formaldehyde fixation of a variety of tissues that are too small, delicate, fibrous, or difficult to see to carry through in situ hybridization in a PCR tube. This method has been used to fix Drosophila spermatocytes and spermatids (both mature and immature), imaginal disks, and malpighian tubules, and whole-mount C elegans after dissection of the gonad. 

There are basically only two protocols that we use for in situ hybridization to whole-mount tissues, and in essence they are identical one is for whole-mount specimens that can be handled in Eppendorf tubes and the other is adapted for whole-mount specimens on slides. As described above, the challenge in extending these methods to new tissues is to find isolation and fixation techniques that enable them to go through one of these procedures. 

Yeast Fixation for in Situ Hybridization and Immunodetection of Nuclear Antigens... 

The first step for a successful in situ hybridization is the fixation of the tissue. This will ensure target nucleic acid retention and preservation of the tissue morphology. Either crosslinking or precipitative fixatives can be used, and a preference for either of the two types of fixative has often been based on the different types of system under investigation (1-7). For hybridization of regulatory peptide mRNA, 4% paraformaldehyde appears to be the most effective, both on tissue blocks and on tissue culture preparations. 

Fixed tissue is generally processed for cryostat sectioning, but in situ hybridization can be equally successful on paraffin embedded tissue (8,9). In any case, it is important always to keep the delay between tissue collection and fixation to a minimum, to avoid nucleic acid degradation, which obviously increases with time delay (4,10). This... 

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