RNA from exposure to RNases present in the antibody and development solutions. To use the same tissue for
in vitro receptor autoradiography and hybridization histochemistry, alternate fresh-frozen sections are used.
Successful hybridization histochemistry seeks a balance between preserving tissue morphology and permeabilizing the tissue to allow access of the probe to the transcripts. While a number of protocols utilize HCl and/or proteases to permeabilize the tissue sections, our approach avoids these harsh treatments through the use of chloroform to de-fat the sections. However, paraffin-embedded tissue sections require the use of protease.
Our experience has shown that the longer riboprobes that, obviously target longer stretches of the transcripts, than single oligodeoxynucleotide probes, offer greater sensitivity. However, the use of multiple oligodeoxynucleotide probes targeted against the same transcript can significantly improve sensitivity compared to a single oligodeoxynucleotide probe. Theoretically, and in practice, riboprobes are more sensitive than the equivalent stretch of bases presented by labeled, double-stranded c
DNA probes. Some researchers also employ alkaline hydrolysis of their riboprobes to increase the ease of tissue penetration. Again, we have not found this to help with our protocol and may be inconsistent in the probe sizes produced. References to other approaches and further discussion are presented at the end of the unit (Valentino et al., 1987; Wilkinson, 1992)
We label our probes for hybridization histochemistry with
35S for a number of reasons. It provides greater resolution and higher efficiency of grain production than either
32P or
33P. Also, it has a half-life of 87 days, compared with 14 and 25 days for
32P or
33P, respectively. These reasons more than compensate for
35S's lower specific activity. Although
3H provides greater resolution and has a much longer half-life, its specific activity is so low, that it is not practical to label probes targeted against transcripts of relatively low abundance. We generally use the digoxigenin-labeled probes to enable double simultaneous detection of two different transcripts within the same tissue sections (and within the same cells). The radiolabeled probes permit more accurate quantitation of transcript levels and are still more sensitive.
Controls for specificity are, of course, the essence of any experiment. Unfortunately, there is no single, absolute control for hybridization histochemistry. Instead, the researcher relies on as many different checks as possible. The ones we prefer, in a roughly descending order of usefulness, are as follows: 1. Same distribution of signal with probe directed against a different portion of the same transcript. 2. Blockage of signal by prior hybridization with unlabeled probe. 3. Correlation of signal with immunocytochemical results. 4. Different distribution of signal with probes against unrelated transcripts, including sense probes (be aware, however, that occasionally the sense probe detects m
RNA transcribed from the opposite
DNA strand. However, a signal with the sense probe does not necessarily invalidate the findings obtained with the antisense probe). 5. Northern analysis using the probe under the same degrees of stringency shows band(s) of expected size(s). Two commonly used controls are not recommended: the use of RNase prior to hybridization is analogous to using a protease prior to immunohistochemistry and the dilution of of labeled probe with unlabeled probe only serves to reduce the specific activity of the probe. Both of these procedures are essentially worthless.
One should be aware of the potential artifacts that arise from autoradiography and/or color techniques. Positive and negative chemography, the spurious creation and destruction of grains, respectively, are constant concerns with autoradiography. Positive chemography is probably more common and is best assessed using sections that were not hybridized, or hybridized with a sense probe. Grains are especially susceptible to loss during staining or after coverslipping if moisture still remains in the tissue sections. These and other aspects of autoradiography are expertly discussed by Rogers (1979). Color development artifacts with alkaline phosphatase may be due to endogenous peripheral-type enzyme and may be blocked with levamisole (intestinal alkaline phosphatase is more refractory and needs treatment with 0.1M HCl for 10 min. at room temperature; Kiyama and Emson, 1991). Also, DTT that is present during the enzymatic development can impart a strong purplish color. The use of non-hybridized sections should reveal whether adventitious color formation is occurring. Loss of alkaline phosphatase staining occurs with exposure to ethanol.
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