A reproducible technique combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine for double labelling studies of cell proliferation in paraffin sections of tissues

Abstract. A method is described to combine tritiated thymidine autoradiography with immunoperoxidase detection of bromodeoxyuridine on the same paraffin sections. It overcomes the varied technical artefacts we encountered when first attempting to combine these techniques and results in preparations with extremely low peroxidase and autoradiographic backgrounds. In particular, we find it is important to avoid the use of detergents during immunostaining, otherwise grain counts are reduced and autoradiograph exposures need to be greatly increased, and to avoid excessive peroxidase staining which makes it difficult to visualize silver grains in the overlying emulsion. The advantages of a method to remove emulsion films using acid-alcohol, allowing the same sections to be dipped twice with a long and a short autoradiographic exposure, are presented. The routine combination of high quality tritiated thymidine autoradiography with clean immunoperoxidase staining of bromodeoxyuridine-positive nuclei provides a new and powerful cell kinetic, double-labelling method to augment existing techniques e.g. by labelling the same cells undergoing DNA synthesis in successive cell cycles.     

A Method to Prevent Wrinkles in Autoradiographic Emulsion when Staining Plastic Embedded Sections with Hematoxylin and Eosin-Phloxine

A procedure was developed which prevents wrinkles in autoradiographic emulsion when sections, embedded in glycol methacrylate, are stained with hematoxylin and eosin-phloxine. Craniofacial tissues labeled with tritiated thymidine were collected and mounted on slides. Slides were dipped in emulsion, stored for one month and developed. The slides were immersed in liquefied celloidin and subsequently stained with a modified hematoxylin and eosin-phloxine procedure. Results showed that the emulsion did not wrinkle and the procedure did not effect the occurrence of labeled cells.     

Factors which Affect Efficiency of Autoradiography with Tritiated Thymidine

The overall efficiency of autoradiography with tritium-labeled thymidine has been found to be influenced by the following conditions: (1) exposure in an atmosphere of CO₂ and the use of the stripping-film technique, both of which increase the autoradiographic efficiency when compared to exposure in air or to dip-coating technique; (2) latent image fading, which increases with increasing exposure. Up to 2 wk of exposure, however, this disadvantage is counterbalanced by the fading of the mechanical background produced during stripping or dip-coating; (3) the thickness of the inert coating interposed between the labeled locus and the sensitized emulsion. A layer of inert coating can be obtained that will arrest all beta particles from tritium, while having no effect on more energetic emitters like C¹⁴; (4) the amount of tritiated thymidine given, with relatively large amounts producing an increase in the mean grain count per labeled cell but not in the percentage of cells identifiable as labeled; and (5) the type of fixative and the staining procedure used. Feulgen stain reduces the mean grain count in cells labeled with radioactive thymidine, while fixation with acetic alcohol (1:3) reduces the grain count in cells labeled with precursors of ribonucleic acid.     

Use of lead aspartate block staining in quantitative EM autoradiography of phospholipids: application to myelinating peripheral nerve

For quantitation of electron microscope (EM) autoradiographs, micrographs must contain clear images which are relatively free of heavy metal precipitates. Satisfactory contrast is usually obtained by staining individual ultra-thin sections with lead citrate. It was recently reported that sequential block staining of tissue with ferrocyanide-reduced osmium tetroxide and lead aspartate produced excellent contrast for EM autoradiography, with sections relatively free of lead precipitate. This protocol avoids the manipulation involved in staining individual ultra-thin sections. We have adapted this method to quantitative EM autoradiographic studies, primarily of phospholipid metabolism in peripheral nerve. We show that block staining with lead aspartate provides: (a) ultrastructural contrast of routinely high quality for myelinated peripheral nerve; (b) high (greater than 98%) retention of glycero-labeled lipid during dehydration and embedment; and (c) a distribution of de novo tritiated glycerol-labeled lipid in ultra-thin sections that is quantitatively identical to the distribution recorded for samples stained by the more laborious post-embedment method. During a 2-hr labeling period in vivo, tritiated glycerol is incorporated into phosphatidylcholine (44%), phosphatidylethanolamine (22%), other phospholipids (16%), and neutral lipids (15%). The analysis of grain distribution in developing sciatic nerve labeled for 2 hr with tritiated glycerol demonstrates that myelinating Schwann cells play the major role in synthesis of endoneurial lipids. Lipid synthesis in myelinated fibers is localized in perinuclear regions of Schwann cell cytoplasm. These regions lie external to compact myelin. Unmyelinated fibers and other endoneurial cells independently incorporate glycerol into lipids.     

A simple method for coupling in situ hybridization and immunocytochemistry: application to the study of peptidergic neurons

We have devised a simple procedure for immunostaining of sections that have previously undergone autoradiographic visualization of mRNAs by in situ hybridization. Classical hybridocytochemistry techniques were performed first on cryostat sections of formaldehyde-fixed tissue. Standard methods were used for slide coating by emulsion dipping and for revelation, fixation, and coverslipping steps. The key to this method is the emulsion removal, or permeabilization, by a short trypsin incubation (0.2% for 20-30 sec) which facilitates the good access of antibodies used in a subsequent immunocytochemical technique to section epitopes. Usual immunofluorescence and immunoperoxidase procedures were successfully performed after this treatment. The immunoreactivity of several neuropeptides was well preserved after this procedure. In addition to its usefulness in our studies, this general method should be applicable to many other situations in which autoradiographic and immunocytochemical detections must be coupled.     

Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data

In order to better characterize the new rapid staining method for flow cytofluorometry proposed by Krishan, we have tested its stability and several other properties, and have carried out a quantitative comparison of the fluorescence histograms obtained using propidium iodide or the acriflavine-Feulgen staining procedure. Using a human hematopoietic cell line in the logarithmic phase of growth, and analyzing the data by means of a mathematical method we have devised, we found that the fluorescence intentsity of cells stained with propidium iodide remains stable for at least 48 h; it is insensitive to dye concentration between 0.025 and 0.10 mg/ml (37-150 muM); it is not affected by incubation with ribonuclease before staining; propidium iodide in 0.1% sodium citrate remains stable for at least 20 days; and quantitative estimates of the fractions of cells in the different phases of the cell cycle are in good agreement with those obtained from acriflavine-Feulgen staining and from autoradiography after pulse labeling with tritiated thymidine. We conclude that this method is useful for the measurement of relative DNA content by flow cytofluorometry, although modifications in the technique are necessary for some cell types which grow in monolayers.     

Tracing neuronal connections with radioisotopes applied extracellularly.

Methodological and technical problems of the autoradiographic-neuroanatomical tracing (ARNT) technique are discussed. The size of the labeled area after a tritiated amino acid injection varies directly with the volume of isotope, the rate of injection, and the length of exposure of the secretion to the emulsion. Frozen sections can be used for autoradiography if they are mounted on subbed slides, dehydrated in ethanol, defatted for 1 hour in xylene, rehydrated through ethanol and water, and dried before coating with emulsion. Brushes used for mounting frozen sections should be used only for this purpose and dipped in boiling distilled water before use to avoid chemoreduced streaks in the emulsion due to contamination from the brushes. Excessive dilution of Kodak emulsion can leave less than a monolayer of grains over certain types of sections; the emulsion thickness should be checked by exposing coated test sections to a brief flash of light and developing immediately. A high intensity safelight recommended for the ARNT darkroom is the Thomas Duplex Super monochromatic sodium vapor bulb safelight; for Kodak NTB-2, -3 and Ilford L4 emulsions the red-banded and yellow-banded filters are used. A useful stain combination for ARNT is Luxol fast blue stained before coating and cresyl violet stained after developing which demonstrates both neuronal cell bodies and myelinated tracts in the same section.     

Double Emulsion Autoradiography for Identifying Tritium-Labelled cells in sections

The sensitivity of tissue autoradiography can be doubled and the number of false negative cells nearly eliminated by interposing thin tissue sections between two layers of photographic emulsion. A mouse was given 50 μc of tritiated thymidine (SA 2,500 c/M) intraperitoneally and killed 1.5 hr later. A portion of the small bowel was removed, fixed and embedded in methacrylate in the usual way. Sections 2 μ thick were cut and allowed to flatten on water at 40° C. Some sections were used to make conventional single emulsion auto-radiographs and other sections were interposed between two layers of emulsion by first coating slides with NTB 3 emulsion, picking up the sections from a water bath at 18° C, drying, soaking 1 min in benzene, drying, and then dipping again in NTB 3 emulsion. They were exposed at 4° C in a low humidity, 100% CO₂ atmosphere for 10 days, developed and covered in the usual way. There was an average of 20.16 ± 1.4 grains per labelled cell in the double emulsion group compared with 10.6 ± 0.9 grains in the single emulsion group. In the double emulsion autoradiographs there were 55.1 ± 1.65 labelled cells per unit area as compared with 39.8 2 2.0 in the single emulsion group.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

We describe a reproducible method for combining tritiated thymidine ([H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [3H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [3H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified. The inhibition of uptake into DNA of [3H]TdR from 0.23 to 1.85 MBq (6.25 to 50 mu Ci) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in mu Ci per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [3H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine [( 3H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12-48 h after [3H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [3H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections. A single peak in the appearance of double-labelled cells was seen at 44 h, if [3H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h. These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44-48 h, but that a few cells cycle more quickly. Double labelling with [3H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

The use of bromodeoxyuridine labeling in the human lymphocyte HGPRT somatic mutation assay

The autoradiographic assay developed by Strauss and Albertini (1979) to quantitate human in vivo somatic mutation at the hypoxanthine guanine phosphoribosyl-transferase locus uses tritiated thymidine to identify mutant cells by their ability to pass through 'S' phase in the presence of 6-thioguanine. An alternative method, based on the incorporation of bromodeoxyuridine (BrdUrd) into the DNA of proliferative cells, followed by differential staining with the fluorescence-plus-Giemsa method, was used to identify 3 classes of lymphocyte nuclei: (a) small darkly stained nuclei, (b) large, reddish-colored nuclei with an apparent nucleolus, and (c) large, bluish-colored nuclei. By double labeling with BrdUrd and tritiated thymidine, it was determined that only the nuclei of the third class had incorporated BrdUrd. These results demonstrate that the technique used for sister-chromatid differentiation can be used to detect putative HGPRT mutants and to determine variant frequencies at the HGPRT locus.     

Fibrillary astrocytes proliferate in response to brain injury: a study combining immunoperoxidase technique for glial fibrillary acidic protein and radioautography of tritiated thymidine.

To determine whether fibrillary astrocytes proliferate in response to brain injury, cells identified as fibrillary astrocytes using immunoperoxidase technique for glial fibrillary acidic protein (GFAP) were examined for uptake of radiolabeled thymidine by autoradiography. In injured mouse brain, autoradiographic label was present over nuclei of immunoreactive fibrillary astrocytes in the lesion site 1 hr following injection of radiolabeled thymidine. The data suggest that fibrillary astrocytes which are sufficiently differentiated to accumulate GFAP retain the capacity to proliferate in response to injury.     

The Application of Autoradiography to Developmental Studies in the Hydrozoa

The uptake of isotopically labelled nucleotides and amino acidshas been studied in five species of hydrozoans. In three speciesthe label was introduced both through immersion in a mediumcontaining the labeled compound and by injecting the labeledcompound into the gastrovascular cavity. In the remaining twospecies the label was introduced by immersion only. The comparisonof soaked and injected specimens clearly indicates that injectionis the method of choice whenever the injection of compoundsinto the gastrovascular cavity is possible. The relative easewith which labeled compounds were absorbed can be correlatedwith the ultrastructure of the epidermal and gastrodermal cellsurfaces and their associated extracellular coats. The use ofthese autoradiographic techniques is illustrated by the useof injected tritiated thymidine and tritiated uiidine to followthe replacement cycle of the zymogenic secretory cell in Hydra,and the use of immersion to introduce tritiated thymidine intothe planula larva of Pennaria.     

A new method for quenching correction leads to revisions of data in receptor autoradiography

Differential quenching of beta-emission affects strongly the analysis of receptor distribution patterns in quantitative receptor autoradiography with tritiated ligands. Different methods for the quenching correction have been described in the past, but some of these are of limited value, if a detailed anatomical parcellation is necessary. Other methods correct exclusively local variations in lipid concentration, which is an important, but only one of several factors causing quenching. A new method for the measurement of quenching (or autoradiographic efficiency) is presented, which permits an anatomically detailed and direct determination of the total quenching without lipid extraction procedures. This method is based on the measurement of autoradiographic efficiency in cryostat sections homogeneously labeled with tritiated formaldehyde by an underlying gelatine section containing this labeled compound. Regional and layer specific measurements of autoradiographic efficiency in cortical and subcortical regions of the human and rat brain are reported. A significant correlation was found between the density of myelin and autoradiographic efficiency but other factors were also shown to influence differential quenching. The use of the here presented correction procedure leads to revisions of the laminar distribution patterns reported for different receptors in human and rat cortical areas. Our results show, that a complete quenching correction is necessary for the mapping of receptor distributions with tritiated ligands.     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Abstract Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine ([³H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12–48 h after [³H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [³H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections.
A single peak in the appearance of double-labelled cells was seen at 44 h, if [³H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h.
These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44–48 h, but that a few cells cycle more quickly. Double labelling with [³H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

Autoradiographic detection of [125I]-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens

We examined whether autoradiographic localization of [125I]-antirabbit immunoglobulin (IgG) was suitable for light and electron microscopic detection of a rabbit antiserum to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), and whether autoradiographic and peroxidase labeling could be combined for simultaneous immunocytochemical identification of TH and neuropeptides in brain. Adult rat brains were fixed by aortic arch perfusion with acrolein and paraformaldehyde. Vibratome sections of the fixed tissues were incubated with various dilutions of TH antiserum followed by [125I]-secondary IgG. These sections were then directly processed for autoradiography or were incubated with rabbit antiserum to substance P (SP) or methionine [Met5]-enkephalin (ME). These latter sections were then processed by the peroxidase-antiperoxidase (PAP) or conjugated peroxidase methods followed by autoradiography. Exposure periods of 12-20 days for light microscopy or 90 days for electron microscopy yielded substantial accumulations of silver grains even at the highest (1:30,000) dilution of TH antiserum. At this dilution, immunoreactivity for TH was virtually nondetectable by PAP and conjugated peroxidase methods. The differential sensitivities of the autoradiographic versus peroxidase methods provided a means for separable identification of rabbit antiserum to TH and to SP or ME. Ultrastructural analysis of the catecholaminergic neurons in the medial nuclei of the solitary tract (NTS) showed selective cytoplasmic localization of silver grains for [125I]-labeling of TH in perikarya, dendrites, and terminals. Within single thin sections prepared for dual labeling, the peroxidase marker for SP and for ME was differentially localized with respect to autoradiographic labeling of TH.     

The cell cycle of glial cells grown in vitro: an immunocytochemical method of analysis.

Studies of cell cycles have traditionally employed [3H]- and [14C]-thymidine to label the DNA of proliferating cells and autoradiography to reveal the thymidine label. The development of antibodies to the thymidine analogue 5-bromodeoxyuridine (BrdU) has allowed the development of an immunocytochemical method analogous to the thymidine autoradiographic technique. In direct comparisons, we found that the immunocytochemical method consistently detected a larger number of proliferating cells. This suggests that it may be a more sensitive index of proliferation than thymidine autoradiography in some systems. We used the BrdU method to analyze the cycle of astroglia cultured from neonatal mouse cerebral cortex. Cells were exposed to BrdU for 1 hr to label a discrete subpopulation of proliferating cells. At 2-36 hr after the pulse, a combination of anti-BrdU immunocytochemistry and counterstaining with propidium iodide was used to identify proliferating cells. The length of the cell cycle was determined by charting the percent of BrdU-labeled mitotic cells vs time after the pulse. We found the average length of the cell cycle of astrocytes grown in vitro to be 20.5 hr. The combined G2 + M phases were 2-3 hr. These values are virtually identical with those found for glial cells in vivo, suggesting that the culture environment does not interfere with the normal control of cell cycle length.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

Abstract We describe a reproducible method for combining tritiated thymidine ([³H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [³H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [³H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified.
The inhibition of uptake into DNA of [³H]TdR from 0.23 to 1.85 MBq (6.25 to 50 μCi) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in μCi per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [³H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

Prevention of Negative Chemography in Phenylenediamine Stained Autoradiographs of Plastic Semithin Sections

Negative chemography is the loss of latent image daring autoradiographic exposure, due to reactive groups in the specimen. Tissue fixed with ghuaraldehyde and osmium tetroxide, block stained with ft-phenylenediamme and embedded in Epou for light microscope sections causes intense negative cbemography when autoradiographed by dipping in Ilford K2 emulsion: this cannot be completely prevented by separating section from emulsion by means of a layer of evaporated carbon. Chemical treatment of the sections before autoradiography may reduce the cbemography. Treatment with 1 % hydrogen peroxide for 15 nun reduced it to such an extent that subsequent coating with 5 nm carbon abolished it. Material block stained in this way gave excellent contrast, both for light and electron microscopy.     

Prevention of negative chemography in phenylenediamine stained autoradiographs of plastic semithin sections

Negative chemography is the loss of latent image during autoradiographic exposure, due to reactive groups in the specimen. Tissue fixed with glutaraldehyde and osmium tetroxide, block stained with p-phenylenediamine and embedded in Epon for light microscope sections causes intense negative chemography when autoradiographed by dipping in Ilford K2 emulsion: this cannot be completely prevented by separating section from emulsion by means of a layer of evaporated carbon. Chemical treatment of the sections before autoradiography may reduce the chemography. Treatment with 1% hydrogen peroxide for 15 min reduced it to such an extent that subsequent coating with 5 nm carbon abolished it. Material block stained in this way gave excellent contrast, both for light and electron microscopy.