Vacuum Freeze Drying of Frozen Sections for Dry-Mounting, High-Resolution Autoradiography

Specimens no larger than 1.5 × 1.5 × 2 mm were frozen in liquid nitrogen and sectioned, while still frozen, with a refrigerated microtome. The frozen sections were dried in a vacuum, then pressed onto either Kodak NTB10 plates or onto slides which had been coated with Kodak NTB3 emulsion and dried. Radioactive mouse liver was used to test tissue preservation. Intestinal mucosa with Ha-labeled nuclei was used to test the quality of autoradiography. Good cytological detail was preserved in both tissues, with the autoradiographs interpretable at the cellular level.     

Effect of Exposure Temperature on Over-All Efficiency of Autoradiographs and on Fogging of Emulsion

Systematic investigations of the influence of various temperatures of exposure on the autoradiographic efficiency and on the rate of fogging of Kodak AR 10 stripping film were carried out. The oxygen content and humidity of the exposed autoradiographs as well as the time of exposure were constant. Exposure of autoradiographs or storage of film at room temperature caused an increase of overall autoradiographic efficiency by approximately 40%, but although the rate of film fogging was also increased, it did not exceed the safe values of background. Both autoradiographic efficiency and rate of fogging were not altered significantly when a temperature of 4 C or -18 C for exposure was used. Therefore, room temperature of exposure is recommended for ordinary autoradiography. For a special autoradiographic technic when low temperature of exposure has to be used, the temperature -18 C may be safely applied without significant impairment of autoradiographic efficiency.     

Growing and Embedding Fern Gametophytes for Autoradiographic Studies

Fern gametophytes were grown in liquid medium on the surface of plastic tissue culture flasks (Falcon Plastics) where they remained attached through radioactive precursor incorporation, fixation in buffered 3% glutaraldehyde, postfixation in buffered 2% OsO₄, alcoholic dehydration, and infiltration with an epoxy resin. Detachment of these plants from the plastic surface occurred only at the final step of infiltration with pure, unpolymerized resin. After detachment, the prothalli were kept in the resin to complete infiltration and then embedded. Sections 1-2 μm thick were cut, floated on a drop of glass-distilled water on clean slides and dried at 70 C. Kodak NTB-2 liquid emulsion was applied to the mounted sections and the emulsion-coated slides stored and developed according to established methods. The resulting autoradiographs showed excellent visualization of reduced silver grains, low background levels, and good preservation of cell structure.     

A Modified Dominici Technic for Autoradiography: Acidified Eosin-Orange and Alcoholic Toluidine Blue

Mouse lymph nodes were fixed in Bouin-Hollande solution, processed through paraffin embedding, sectioned and coated with Kodak NTB3 liquid emulsion. After development, the autoradiographs were stained 10 min in a 0.5% eosin Y-orange G mixture acidified to pH 4.4, differentiated in 0.5% acid alcohol and counterstained 10 sec with 0.25% toluidine blue O in 50% ethanol. The technic yielded distinct differentiation of neutrophils, eosinophils, blast cells, plasmacytes, mast cells, macrophages, reticular cells, histiocytes, lymphocytes and other lymphoid cells. The method also provided sensitive definition of the cytoplasmic and nuclear characteristics of all cell types.     

QUANTITATIVE TRITIUM AUTORADIOGRAPHY OF MAMMALIAN CHROMOSOMES : I. The Basic Method

A technique has been developed that allows repeated autoradiographs to be made of the isotope distribution in the chromosomes of a single cell. A series of 10 separate autoradiographs were made of a Chinese hamster diploid male metaphase cell which had been labeled with tritiated thymidine during the first 15 minutes of its DNA synthesis period in the previous interphase. Each autoradiograph had low grain densities above the chromosomes so that quantitation was feasible. The separate autoradiographs were photographically combined into a single composite in which grain images were converted to lines oriented at right angles to the chromosome axis. The line densities were then measured with a recording microdensitometer to yield graphs reflecting the isotope distribution along each chromosome. The area under each graph was directly proportional to the total number of grains counted above the corresponding chromosome in the 10 separate autoradiographs. The distribution of isotope along the chromosomes was different for each chromosome, and in some cases homologs also differed in their early labeling patterns.     

Autoradiography and epifluorescence microscopy combined for the determination of number and spectrum of actively metabolizing bacteria in natural water.

A technique is described for the determination of bacterial numbers and the spectrum of actively metabolizing cells on the same microscopic preparation by a combined autoradiography/epifluorescence microscopy technique. Natural bacterial populations incubated with [(3)H]glucose were filtered onto 0.2-mum Nuclepore polycarbonate membranes. The filters were cut into quarters and fixed on the surface of glass slides, coated with NTB-2 nuclear track emulsion (Kodak), and exposed to the radiation. After processing, the autoradiographs were stained with acridine orange. A combination of overstaining on the slightly alkaline side and gradual destaining on the acid side of neutrality gave the best results. Epifluorescence microscopy revealed bright-orange fluorescent cells with dark-silver grains associated against a greenish-to-grayish background. Based on the standardization curves, detection of actually metabolizing cells was optimal when cells were incubated with 1 to 5 muCi of [(3)H]glucose per ml of sample for 4 h and the autoradiographs were exposed to NTB-2 emulsion at 7 degrees C for 3 days. In water samples taken immediately above sandy sediments at beaches of the Kiel Fjord and the Kiel Bight (Baltic Sea, FRG), between 2.3 and 56.2% (average, 31.3%) of the total number of bacteria were actually metabolizing cells. Spearman rank correlation analysis revealed significant interrelationships between the number of active bacteria and the actual uptake rate of glucose.     

INCORPORATION OF H3-THYMIDINE INTO SHOOT AND ROOT APICES OF CERATOPTERIS THALICTROIDES

Gifford , Ernest M., Jr . (U. California, Davis.) Incorporation of H³-thymidine into shoot and root apices of Ceratopteris thalictroides. Amer. Jour. Bot 47(10): 834–837. Illus. 1960.—The localization of tritiated thymidine in apical meristems of Ceratopteris thalictroides by the autoradiographic method is described. Intact, floating plants of the fern were placed in 1/2 strength Hoagland's inorganic nutrient solution containing H³-thymidine (10 μc/ml.) for 3 days. The material was killed, dehydrated and embedded in paraffin. Autoradiographic stripping film (AR 10 Kodak) was applied to serial sections. After an appropriate exposure period, the film was developed and the sections with the superimposed film were stained lightly with Harris' hematoxylin. The autoradiographs revealed the presence of the H³-thymidine in nuclei of the large, individualized apical cells of shoots and roots which is proof of DNA synthesis. In no instances were these nuclei unlabeled. If endomitotic reduplication is excluded the results of these studies lend support to the concept that apical cells actually do divide and perhaps at a higher rate than envisioned by other workers. Considerable cytoplasmic labeling occurred and its significance to general problems of DNA synthesis is discussed.     

Wire-Loop Application of Liquid Emulsion to Slides for Autoradiography in Light Microscopy

This is an adaptation of procedures used to prepare autoradiographs for electron microscopy to light microscopy. A rectangular wire loop slightly larger than the slide is used. The liquid emulsion is prepared by mixing 1 volume of a 0.1% solution of Dreft or Ivory Snow with 2 volumes of Kodak NTB3 nuclear track emulsion. The slide to be coated is placed on a cork stopper supported by a glass plate 61 cm from the safelight. The loop was withdrawn from the liquid emulsion, with the plane of the loop parallel to the surface of the liquid, placed directly over the slide and slowly lowered so that the film (in the sol state) was broken by the slide. If desired, the film can be allowed to gel before application to the slide by waiting approximately 40 sec after the loop is removed from the liquid emulsion. That the developed emulsion is consistently uniform was indicated by a thickness of 0.4 ± 0.02 μm. Diluted emulsion can be stored after use for 1-2 wk at 2-8 C and used a second time before discarding. The identification of human D group chromosomes labeled with tritiated thymidine as well as prescreening patients for the Lesch-Nyhan syndrome, which utilizes tritiated hypoxanthine labeling, have been successfully carried out by applying the emulsion in the sol state.     

Autoradiography and Epifluorescence Microscopy Combined for the Determination of Number and Spectrum of Actively Metabolizing Bacteria in Natural Waters

A technique is described for the determination of bacterial numbers and the spectrum of actively metabolizing cells on the same microscopic preparation by a combined autoradiography/epifluorescence microscopy technique. Natural bacterial populations incubated with [³H]glucose were filtered onto 0.2-μm Nuclepore polycarbonate membranes. The filters were cut into quarters and fixed on the surface of glass slides, coated with NTB-2 nuclear track emulsion (Kodak), and exposed to the radiation. After processing, the autoradiographs were stained with acridine orange. A combination of overstaining on the slightly alkaline side and gradual destaining on the acid side of neutrality gave the best results. Epifluorescence microscopy revealed bright-orange fluorescent cells with dark-silver grains associated against a greenish-to-grayish background. Based on the standardization curves, detection of actually metabolizing cells was optimal when cells were incubated with 1 to 5 μCi of [³H]glucose per ml of sample for 4 h and the autoradiographs were exposed to NTB-2 emulsion at 7°C for 3 days. In water samples taken immediately above sandy sediments at beaches of the Kiel Fjord and the Kiel Bight (Baltic Sea, FRG), between 2.3 and 56.2% (average, 31.3%) of the total number of bacteria were actually metabolizing cells. Spearman rank correlation analysis revealed significant interrelationships between the number of active bacteria and the actual uptake rate of glucose.     

A STAINING TECHNIQUE FOR AUTORADIOGRAPHS OF NONDIFFUSIBLE ISOTOPES

Nondeparaffinized radioactive tissue sections are stained with hematoxylin and eosin by being floated on aqueous solutions for 1 hr each. The sections are then thoroughly washed, dried and exposed to autoradiographic plates or emulsions for predetermined periods of time. When desirable, both stained and unstained adjacent tissue sections can be mounted on a single slide of autoradiographic plate for exposure. Kodak DK-19 and 30% Na₂S₂O₃.5H₂O solutions are used for subsequent developing and fixing. The finished autoradiographs show excellent resolution and cytologic detail, since the gelatin remains unstained while the tissue retains its stain. Stains other than hematoxylin and eosin can be applied to this technique, provided they withstand the developmental and fixation processes.     

A staining procedure for identifying viable cell hybrids constructed by somatic cell fusion, cybridization, or nuclear transplantation

A general procedure for identifying viable hybrid cells was developed. One cell type was labeled by a brief incubation in the Kodak laser dye rhodamine 123, which accumulates in the mitochondria; a second cell type was labeled by a brief incubation in the Hoechst fluorochrome 33258, which binds to chromatin. The substances which are eventually lost from the organelles, appeared to be nontoxic; the plating efficiencies of numerous cell lines tested was unaffected. Either whole cells or cytoplasts labeled with rhodomine 123 were fused, using inactivated Sendai virus, to whole cells or karyoplasts labeled with Hoechst 33258. When living cells were illuminated with ultraviolet light, individual whole cell hybrids, cybrids or cytoplasmic- nuclear hybrid cells could be rapidly identified by the appropriate staining pattern.     

The distribution of 14c from [U-14c]glucose in mice using whole-body autoradiography.

Tissue distribution of radioactive carbon from [U-14C]glucose in the mouse in vivo was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-acetone at 0.5, 1, 5 and 30 min, 1 and 24 hr and 1 and 3 weeks after intraperitoneal injection of [U-14C]glucose. Whole-sagittal sections of the frozen mouse, obtained by using a microtome in a cryostat, were dried in a cryostat and autoradiographed. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HClO4, dried at room temperature and again autoradiographed. Autoradiographs that have undergone this process are referred to as treated autoradiographs. In both untreated and treated autoradiographs, within 1 min following injection of the labeled glucose, the abdominal cavity had the highest autoradiographic density. At 1 hr, density became highest in Harder's, sublingual and duodenal glands, large intestinal mucosa and tongue, and after 3 weeks, no autoradiographic denisty was present.     

Making Autoradiographs of Individual Blood Cells

Single blood cell autoradiographs were made by smearing blood, diluted with serum, directly upon the emulsion of an Eastman NTB plate. After exposure, the plates were developed in D19. A 10% sodium sulfate solution was used for the photographic fixing to prevent lysis of the cells. After staining with Wright's stain the cells and autographs could be examined simultaneously under the microscope.     

A Technique for Embedding Undecalcified Bone Samples for Detecting Alpha-Emitters Using Vacuum Impregnation with Spurr's Resin

A method has been developed by which large samples of mineralized bone, containing an alpha-emitter, can be embedded in Spurr's resin in a fraction of the time required by conventional methods. Bone samples were freeze-dried or fixed and dried prior to impregnation with Spurr's resin under vacuum. Sections were cut for the preparation of either alpha-track or fission-track autoradiographs using the solid state detector CR-39. This method is applicable to samples containing a mobile form of a radionuclide that may be translocated during the processes of fixation and dehydration of the specimen.     

The use of microcomputers for the quantitation of light intensity patterns using digitized video signals

We have developed an inexpensive yet versatile microcomputer-basedsystem for quantitating light intensity levels in autoradiographs.This system employs a standard video camera interfaced to ananalog-to-digital convertor. A program has been written forthis system which can measure intensities within a defined regionof an autoradiograph, permitting an easy and accurate quantitationof spots or bands of irregular shape. Received on June 18, 1985; accepted on September 3, 1985     

Absence of oxytocin-neurophysin messenger RNA in the day-18 bovine conceptus

Total cellular RNA was isolated from conceptus tissue obtained from 22 superovulated cows 18 days after artificial insemination. Total RNA was also isolated from luteal tissue from 3 cyclic cows 7 and 8 days after oestrus. Luteal and conceptus RNA were simultaneously subjected to formaldehyde-agarose gel electrophoresis and transferred to nitrocellulose by bidirectional diffusion blotting. Northern blots were probed using cDNAs specific for bovine oxytocin and bovine beta-actin gene sequences. Hybridization of the oxytocin cDNA to RNA was consistently observed on autoradiographs as a 0.6 kilobase (kb) band in lanes containing corpus luteum RNA, but was not detected in lanes containing conceptus RNA. The presence of conceptus RNA on the blots was confirmed by hybridization of the actin cDNA to conceptus RNA, which resulted in a 2.0 kb band on autoradiographs. These results suggest that oxytocin is not synthesized by the bovine conceptus on Day 18 of gestation.     

Resolution of electron microscope autoradiography. IV. Application to analysis of autoradiographs

The previous publications of this series described the expected grain distributions around model radioactive structures in EM autoradiographs as a function of the specimen resolution. This family of expected distributions was called the "universal curves". In the present study, experiments on 14C-sources were compared, significant differences were found depending on the energy of the isotope. These differences were primarily in the tails of the distributions, and are therefore important in correcting for cross-scatter when analyzing electron microscope autoradiographs. Using the universal curves unique for 125I, 3H, and 14C, we designed three sets of transparent overlays, or "masks", one set for each of these isotopes. The masks can be used by an investigator in a manner similar to that suggested by Blackett and Parry to generate grain distributions in autoradiographs on the basis of any desired hypothesis regarding the levels of radioactivity in different structures. A subsequent comparison between these generated distributions and those obtained from the observed grains in these autoradiographs leads to a determination of the most likely levels of radioactivity in the tissue. A computer (described in an Appendix by Land and Salpeter) can be used to find the "best fit" levels of radioactivity in complex cases. The accuracy of the masks was checked on generated line sources for each of the three isotopes.     

A blood analog for laser-induced photochemical anemometry

A transparent viscoelastic blood analog fluid was developed for use with Laser-Induced Photochemical Anemometry. To provide solubility of the photochemical tracer, 1′, 3′, 3′-trimethyl-6-nitroindoline-2-spiro-2-benzopyran (TNSB dye, Kodak Chemicals), the analog solvent needed to be nonpolar, thus currently available aqueous blood analogs were not suitable. An analog consisting of 0.04% ethylhydroxyethylcellulose dissolved in gamma-butyrolactone produced a pseudoplastic steady shear response with low elasticity in unsteady shear, while being compatible with the photochemical tracer.     

ELECTRON MICROSCOPIC AUTORADIOGRAPHY : An Improved Technique for Producing Thin Films and Its Application to H3-Thymidine-Labeled Maize Nuclei

An improved method has been developed for the preparation of autoradiographs for electron optical study. The refinement lies principally in the routine production of uniformly thin layers of photographic emulsion over the tracer-labeled cell sections. This is accomplished by means of a centrifugal spreading mechanism. Maize root tips grown in the presence of H³-thymidine were examined electron microscopically by this technique and were found to display nuclear labeling with impressive clarity. The new procedure utilized here yields objects in which the finest cell structures are easily resolvable without recourse to gelatin digestion techniques.     

Studies on regional blood flow of the mouse using whole-body autoradiography of 14C-iodoantipyrine

Summary In order to visualize regional blood flow in various tissues of the mouse at the same time, the distribution of radioactive carbon from ¹⁴C-iodoantipyrine was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-hexane at 1, 10, 30 min, and 1 h and 3 h after intravenous injection of ¹⁴C-iodoantipyrine. Whole-sagittal sections of the frozen mouse, obtained by using a cryostat microtome, were dried in a cryostat and subjected to autoradiography. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HClO₄, dried at room temperature and again subjected to autoradiography. Autoradiographs thus obtained are referred to as treated autoradiographs. It was found that the method could be suitable for the estimation of regional blood flow of the renal cortex, spleen, lung, skeletal muscle, bone marrow, thymus, testes, and brain.