A laser densitometer for selective spot analysis on dot blots and two-dimensional gel autoradiograms

We describe an inexpensive densitometer, employing a small HeNe laser and an IBM-compatible personal computer that performs accurate measurements of selected spots on two-dimensional gel autoradiograms or chromatograms with an accuracy and a sensitivity equal or superior to those of many commercial instruments. Our open-table design allows the operator to visually monitor the scanning process in room lighting, and provides great flexibility in both the size and the nature of items to be scanned. The instrument has two moving parts (a prism and a small motor). A commercially available software package (ASYST) acquires digital data, graphs the data on the TV monitor, converts the data to optical density or to radioactive incorporation (cpm), subtracts background, integrates peak areas, and stores data on disk. The total time for these operations is 20-30 s per spot. The instrument has a dynamic range of 0.25 to 3.0 OD units and can measure a 10,000-fold range of 14C or 35S isotope concentrations on autoradiograms. The complete device can be assembled with a hobbyist's knowledge of electronics, moderate programming abilities (no machine language required), and a cost of less than $3000, not including the IBM PC.     

Quantitative autoradiography of neurochemicals

Several new methods have been developed that apply quantitative autoradiography to neurochemistry. These methods are derived from the 2-deoxyglucose (2DG) technique of Sokoloff (1), which uses quantitative autoradiography to measure the rate of glucose utilization in brain structures. The new methods allow the measurement of the rate of cerbral protein synthesis and the levels of particular neurotransmitter receptors by quantitative autoradiography. As with the 2DG method, the new techniques can measure molecular levels in micron-sized brain structures; and can be used in conjuction with computerized systems of image processing. It is possible that many neurochemical measurements could be made by computerized analysis of quantitative autoradiograms.     

In situ dot blots: quantitation of mRNA in intact cells.

A rapid, simple and reproducible dot blot method is described for quantitating the amounts of specific messages in small numbers of intact cells. The method has been used to accurately determine the number of histone H4 mRNA molecules in growing (approximately 40,000) and in starved (approximately 1600) Tetrahymena thermophila, and to measure the amount of message contributed by an E. coli plasmid containing part of the S10 ribosomal operon. Use of the method is illustrated to optimize in situ hybridization protocols and to measure mRNA amounts in cell lysates. Preliminary studies also indicate that the method can be used to detect mRNA in intact yeast cells.     

Study of X chromosome abnormality in XX males using bivariate flow karyotype analysis and flow sorted dot blots

We have used bivariate flow karyotype analysis to quantify aberrant X chromosome size in 11 XX males. With one exception, the patients could be grouped into those with an X homologue difference greater than normal (Group A, n = 3) and into those whose X homologue difference could not be distinguished from female controls (Group B, n = 7). The range of sizes of the aberrant X chromosome in Y-sequence positive patients agrees with the variable nature of the X-Y interchange in these individuals as determined by the use of Y-specific DNA probes and Southern blotting analysis. In one patient it was possible to sort separately the normal and the X-Y interchanged homologues for dot blot analysis. The presence of Y sequences and an increased dose of the zinc finger gene, ZFY, were detected in the X-Y interchanged homologue. In preliminary studies of 5 male and 6 female controls, it was noted that a consistent difference between the two X homologues in females was found which could not be totally explained by errors of the fitting procedure. We suggest that this difference could be due to X inactivation and that the two X homologues in females might be distinguishable.     

Quantitative tritium autoradiography of mammalian chromosomes

The rates of tritiated thymidine accumulation in each of the chromosome types in Chinese hamster line Don and strain Don-C have been assayed by quantitative tritium autoradiography. The late-replicating nature of the X and Y chromosomes is readily apparent. Many chromosomes exhibit three separate steps of synthesis, with reduced rates of thymidine incorporation between 3 and 4 hours and again between 5 and 6 hours. The same three-step pattern can be seen in scintillation data from FUdR synchronized cells, with 40% of the DNA made in each of the first two steps and 20% in the final step.This research was supported in part by Grant GB-7248 from the National Science Foundation and by Grant E-286 from The American Cancer Society.     

A spectrophotometric microassay for sulfated glycosaminoglycans using a laser densitometer

The absorption spectrum of the dye 1,9-dimethylmethylene blue shifts if complexed with sulfated glycosaminoglycans. The present method uses the decrease in A633 rather than the increase in A535, described in a recent method, to measure the sulfated glycosaminoglycan content of biological samples. A conventional spectrophotometer was used to estimate the levels of sulfated glycosaminoglycan in papain extracts from intestinal wall tissue, by measuring both the A535 and the A633 and comparing them with a chondroitin sulfate standard: a highly significant correlation (r = 0.974, n = 17) was obtained. Also, interference by substances like RNA, DNA, and hyaluronic acid was similar for both methods. These results allowed us to employ a laser densitometer with a helium/neon laser emitting at 633 nm to improve the sensitivity and the capacity of the assay. The combination of a small reaction volume and a high-intensity light source allows the detection of less than 0.1 microgram chondroitin sulfate, a 40-fold improvement in sensitivity as compared with the original method. A very significant correlation (r = 0.885, n = 17) existed between results obtained with the macroassay, using a spectrophotometer, and those found by employing the microassay, using the laser densitometer. The use of microtiter plates and the screening potential of the densitometer yields an assay which is fast, very sensitive, and suitable for processing large numbers of samples.     

Long-lived southern blots using RNA probes

Conditions for preparation and hybridization of Southern blots are described which assure reusability through 15 to 25 cycles. The procedure relies on the use of charge-modified nylon membranes and^[32P]-labelled RNA probes.     

Quantitative multiplexed quantum dot immunohistochemistry

Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8 h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.     

Quantitative receptor autoradiography in the human brain

Summary Quantitative receptor autoradiography on sections of the human brain raises methodical problems of which some are relevant also for studies in animal tissue, but others are unique in studies of human brain tissue. Procedures for the following methodical aspects are discussed image analysis for quantitation of the regional distribution of receptor densities, saturation analysis on autoradiographs, influence of age and post-mortem delay and quenching of -radiation in brain tissue. The solutions proposed to these problems make receptor autoradiography in the human brain to a reliable method for studies of chemical neuroanatomy.     

An automated analysis of chemotaxis in vitro using a computer-assisted scanning densitometer

The quantitation of chemotaxis in vitro was developed with a computer-assisted scanning densitometer. The method of estimating the number of cells on a filter was based on the photo-reflection from the nuclei of stained cells. Samples obtained from a 48-well micro chemotaxis assembly were successfully analyzed by this method. This assay system could quantitate chemotaxis much faster and more accurately than by cell counting under the microscope. It was sensitive enough to determine the responsiveness of SMCs and fibroblasts to various chemoattractants. This system could be applied to medical and biological screening tests for drugs and clones in laboratories.     

Western blots using stained protein gels.

A general method is described for the electrophoretic transfer of proteins from stained gels to membranes and subsequent Western detection of specific proteins on the stained membranes. Proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gels are stained using either of two different methods followed by electrophoretic transfer to nitrocellulose or Immobilon-P membranes. The transferred proteins remain stained during immunodetection, providing a set of background markers for protein location and size determination.     

DNA-binding domains of fibronectin probed using Western blots

Human plasma fibronectin was subjected to limited proteolysis, and its DNA-reactive polypeptides were distinguished using the protein blot technique. The polypeptides were separated electrophoretically by SDS gel electrophoresis, transferred to nitrocellulose and probed with radiolabeled human lymphocyte DNA. The major DNA-binding domains identified by the protein blots were comparable to the polypeptides identified using affinity chromatography on DNA-cellulose.     

Enzyme activity dot blots: a rapid and convenient assay for acetyltransferase or protein kinase activity immobilized on nitrocellulose

Methods are described for assaying (Tetrahymena) histone acetyltransferase activity and (Drosophila) casein kinase II activity by spotting extracts on nitrocellulose filters. The methods are quantitative over a wide range of enzyme concentrations and are almost as sensitive as liquid assays. Examples are presented for illustrating the use of these methods for enzyme purification, concentration, and desalting, as well as for electrophoretic blotting from agarose gels. A simple method for autoradiographic enhancement of nitrocellulose filters is also described.     

Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera.

Digital chemiluminescence imaging with a cryogenically cooled charge-coupled device (CCD) camera is used to visualize DNA sequencing fragments covalently bound to a blotting membrane. The detection is based on DNA hybridization with an alkaline phosphatase(AP) labeled oligodeoxyribonucleotide probe and AP triggered chemiluminescence of the substrate 3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD). The detection using a direct AP-oligonucleotide conjugate is compared to the secondary detection of biotinylated oligonucleotides with respect to their sensitivity and nonspecific binding to the nylon membrane by quantitative imaging. Using the direct oligonucleotide-AP conjugate as a hybridization probe, sub-attomol (0.5 pg of 2.7 kb pUC plasmid DNA) quantities of membrane bound DNA are detectable with 30 min CCD exposures. Detection using the biotinylated probe in combination with streptavidin-AP was found to be background limited by nonspecific binding of streptavidin-AP and the oligo(biotin-11-dUTP) label in equal proportions. In contrast, the nonspecific background of AP-labeled oligonucleotide is indistinguishable from that seen with 5'-32P-label, in that respect making AP an ideal enzymatic label. The effect of hybridization time, probe concentration, and presence of luminescence enhancers on the detection of plasmid DNA were investigated.