Vitro Labeling Autoradiography

As mentioned above, the in vivo labeling approach was limited to the study of only a few receptors. Also, because one cannot control various aspects of the labeling procedure, such as the concentration of drug at receptor improved methods for labeling receptors were needed. Accordingly, we devised an in vitro labeling procedure whereby slide-mounted 

TISSUE SECTION LABELING ON SUBBED INCUBATION SLIDE 1+ DISPLACER  

SUBBED SLIDE WITH RECEPTOR LABELED TISSUE 

Advantages of in Vitro Labeling Autoradiography over in Vivo Labeling Autoradiography 

Enhanced sensitivity (nanogram-size regions studied quantitatively) 

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Deductions concerning the loci of proteoglycan biosynthesis come om a considerable amount of work involving radioactive labeling of proteoglycans in vitro and autoradiography of the labeled tissue, and from investigation of a number of induced effects upon biosynthesis— some of such work has been reviewed (S46). 

Kuhar M J and Unnerstall J R (1982) In vitro labeling receptor autoradiography loss of label during ethanol dehydration and preparative procedures Brain Res 244, 178-181... 

In order to have a more convenient synthesis of a MAO B-selective [ Fj-labeled inhibitor, Mukherjee and coworkers employed a similar strategy to the one they used to make [ Fj-labeled clorgyline. The synthesis of [ Fj-labeled A/-(6-fluorohexyl)-A/-methylpropargylamine (39) is shown in Scheme 2. Binding of 39 had over 200-fold selectivity for MAO B over MAO A. In vitro autoradiography of rat brain slices showed that 39 accumulated in regions known to have high concentrations of MAO B activity [107]. 

Fig. 1. End labeling of eubacterial RNA with [a-MP]GTP in vitro. Deproteinized RNA preparations were incubated for 1 hr at 43 (lane 1) and 50° (lane 2) with (a-32P]GTP as described, then fractionated on a 5% polyacrylamide-8 M urea gel. Labeled fragments were visualized by autoradiography. Lane 1, Agrobacterium tumefaciens A136 lane 2, Azoarcus sp. BH72. Scale at right indicates position of Haelll restriction fragments of phage 4>X174 DNA in nucleotides.

Liver cells are then exposed in vitro to the test compound and incubated with tritium-labeled thymidine for about 18 hours. At the end of the incubation, the cells are fixed on slides and prepared for autoradiography. For that the slides are first exposed to liquid photographic emulsion, air-dried and following a 7-day exposure in the dark, exposed to developing solution. 

Study shows that there are no adverse effects after administration of the material at the limit (high) dose generally used in the pharmaceutical industry. An investigative mass, balance, whole-body autoradiography study provides information on absorption, distribution, metabolism, and excretion. This study also involves an investigation of suitable labeling of the material. An in vitro metabolism study (e.g., in rat vs. human hepatocytes) may also be useful for modified food additives and excipients to compare the break-down process and to show possible differences between rat and human in these processes. 

Other possibilities are the radioactive labeling in vitro of one of the monoclonal antibodies and enzyme-labeling of the other and their detection in cells by autoradiography (Tijssen and Kurstak, 1977) or the complexing of heavy metals to one of the immunoreact-ants (e.g., colloidal gold to SpA Roth, 1982). 

In vitro translations were carried out according to the manufacturer (Promega Biotech) with [35S] methionine (1000 Ci/ml). After 1 hour incubation at 30 C, 1 jul-aliquots of the translation mixtures were used to determine hot trichloroacetic acid (TCA) precipitable incorporation of [35S] methionine. Products were analized by SDS-electrophoresis (SDS PAGE)(4). Gels were stained and dried, and labelled bands were detected by autoradiography. 

The principle in vitro types of application are for radioimmuno- (or related) assay and pre-clinical development studies prior to future in vivo use, but antibodies may also be labelled for use as probes in immunocytochemistiy. Western blots, or for immunoprecipitation. The choice of radionuclide will depend upon such factors as ease of tracer preparation, ease and efficiency of detection (counting or autoradiography), shelf-life of radiotracer, cost, and safety. A list of some of the most common radionuclides which might be employed for preparation of radiotracers with applications in vitro, together with their physical decay properties is shown in Table 1. 

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