Quantitative immunofluorescence

Summary Instrumental requirements for quantitative immunofluorescence were analyzed. Commercially available equipment was found to be sufficiently sensitive and light sources such as a halogen lamp and the 75 W xenon high pressure arc lamp were found not to fluctuate more than one percent if operated on a stabilized power supply. Only minor modifications of the instrumentation were needed to obtain readings with a reproducibility within one percent, provided the environmental temperature was kept constant. A uranyl glass slide proved to be a suitable calibration standard, to be used for each series of measurements. As standard fluorescein unit (SFU) 10^–3 of the fluorescence of 1 picoliter of a 50 M fluorescein solution pH 8.5 was chosen. The pH of fluorescein dyes had to be about 8.5 since the fluorescence was then maximal and the influence of small pH variations neglectable. The SFU was measured either from microdroplets of fluorescein solution or in microcapillaries containing this solution. The microcapillary system is to be preferred since it is easier to handle, measurements are less time consuming and fading is less disturbing the measurements.From the Study Group on Quantitative Immunofluorescence.     

Quantitative Autonomic Testing

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.     

Quantitative plant proteomics

Quantitation is an inherent requirement in comparative proteomics and there is no exception to this for plant proteomics. Quantitative proteomics has high demands on the experimental workflow, requiring a thorough design and often a complex multi-step structure. It has to include sufficient numbers of biological and technical replicates and methods that are able to facilitate a quantitative signal read-out. Quantitative plant proteomics in particular poses many additional challenges but because of the nature of plants it also offers some potential advantages. In general, analysis of plants has been less prominent in proteomics. Low protein concentration, difficulties in protein extraction, genome multiploidy, high Rubisco abundance in green tissue, and an absence of well-annotated and completed genome sequences are some of the main challenges in plant proteomics. However, the latter is now changing with several genomes emerging for model plants and crops such as potato, tomato, soybean, rice, maize and barley. This review discusses the current status in quantitative plant proteomics (MS-based and non-MS-based) and its challenges and potentials. Both relative and absolute quantitation methods in plant proteomics from DIGE to MS-based analysis after isotope labeling and label-free quantitation are described and illustrated by published studies. In particular, we describe plant-specific quantitative methods such as metabolic labeling methods that can take full advantage of plant metabolism and culture practices, and discuss other potential advantages and challenges that may arise from the unique properties of plants.     

Quantitative succinate-dehydrogenase histochemistry

Summary In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are choosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.     

Quantitative Clinical Pathology

Edited by Peter Hamilton and Derek Allen. 1995. Blackwell Science, Oxford. 342 pages. Hardback. ISBN 0-632-03286-3.     

Quantitative footprinting analysis

This review outlines the steps for obtaining relative constants for drugs from footprinting data. After correcting the autoradiographic spot intensities for differing amounts of radioactive DNA loaded into the lanes of a sequencing gel, footprinting plots, showing individual spot intensities as a function of drug concentration, are constructed. The initial relative slopes of footprinting plots are proportional to the binding constant of the drug for its DNA sites. Slopes of plots outside the drug binding sites can be used to identify locations of altered DNA structure. It illustrates the power of quantitative footprinting analysis by analyzing the binding of the antiviral agent netrospin to a 139-base pair restriction fragment in the presence of the antitumor agent actinomycin D. While two netrospin binding regions are unaffected by actinomycin D a third region experiences enhanced binding in the presence of the antitumor agent.     

Quantitative complement fixation

Summary In agreement with the investigations ofKent et al. (4), it was found that blood collected in a modifiedAlsevers solution remains constant for a period of at least 14 days.We also succeeded in proving the same with regard to complement, stored according toRichardson (9) and with regard to amboceptor prepared according to the directions of the New York State Department of Health and stored in an equal volume of glycerol at 4° C.The cheap Eel colorimeter proved very suitable for the standardization of the erythrocyte-suspension and the measurement of the percentage of haemolysis.Success was not obtained by means of the method, used in this investigation, to keep the amount of complement, which under the conditions of the test causes haemolysis of 50% of erythrocytes, so constant, as to make daily complement titration unnecessary.