Factors which Influence the Latent Image in Autoradiography

Sections treated with N-ethyl-maleim'ide, a sulfhydryl blocking reagent, exhibit similar grain counts over nonradioactive and S-labeled tissues. This reveals that no chemo-graphic effects were produced by sulfhydryl groups in tissues fixed and prepared in the manner described. It was noted, however, that background count over tissue sections is consistently tower than over adjacent him, indicating perhaps the existence of substances in tissues which interfere with the production of the latent image. These effects have been eliminated by coating the tissues with celloidin. Celloidin coating can be used for most emitters including S but not for tritium. A special method for tritium, not involving celloidin coating is given.     

Celloidin as a protective film for improving the histochemical localization of succinate dehydrogenase

Synopsis The effect on the localization of succinate dehydrogenase of coating fresh and frozen-dried cryostat sections of unfixed hamster liver with celloidin was examined. It was found that the protective celloidin film not only leads to a more discrete localization of the enzyme, but is also prevents high losses of nitrogenous materials from sections into the incubation medium, a 30% loss in contrast to the 70% with fresh, non-coated sections. Further, after incubation, the morphology of the coated sections is much better than the uncoated ones.     

A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.

The methods for sectioning, and the removal of the embedding agent are given.

The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.     

Nongraded Dehydration and Low-Pressure Infiltration for Rapid Celloidin Embedding of Brain Tissue

Various ways of shortening single steps in the celloidin process have been combined to form a routine method which may be completed, for tissues of average size, within a week following fixation. Fixed, washed tissue slices 5 mm thick are dehydrated in 1 or 2 changes of absolute ethanol and acetone, 1:1. This requires 24 hr in an incubator at 37 C, or 12-16 hr if a magnetic stirrer is used. After ether-alcohol for 4 hr. the tissues are transferred to 5% celloidin and infiltrated in a vacuum desiccator attached to a filter pump. When the volume of celloidin is reduced to half the original amount (about 2 hr), the tissues are removed from the infiltrating fluid and embedded in 10% celloidin. The blocks are hardened in chloroform and cleared by suspending them in 2 or 3 changes of terpineol agitated by a magnetic stirrer. Sections are cut in terpineol, using any type of microtome. After washing in 95% alcohol, they are mounted on albumenized slides for staining.     

Pasternack's Paraffin Method Modified for Plant Tissue

This method for preparing paraffin sections of plant material is a modification of Pasternack's one-hour method for animal tissues. Fixation in Randolph's CRAF fixative is hastened by heat and increased vapor pressure obtained by the use of screw top vials. Dehydration with Zirkle's butyl alcohol series likewise is hastened in the same manner. The rapid penetration of paraffin by the use of 1/2 paraffin and 1/2 butyl alcohol in heated screw top vials shortens the embedding process. Sections are held on the slide thru staining by albumen fixative and a coating of 0.2% celloidin in absolute alcohol and ether. Good penetration with freedom from shrinkage or distortion is obtained and root tip chromosome counts can be made in approximately 3 hours.     

Deplasticizing of thick Epon sections involving a new adhesive technique and staining of nervous tissue

The present communication deals with a technique developed for the selective staining of neural tissue in thick (10 micron) Epon sections. A new adhesive method was needed, because the known techniques are only applicable to 0.5-2 micron thin sections. The critical step in the procedure is the adhesion of the sections onto the slides. This is accomplished by heating the sections on top of a uniform layer of albumin glycerol on the slide followed by coating with celloidin. The results after deplasticizing and coagulation with this technique are comparable to those obtained by paraffin or frozen section techniques, but in addition have the advantage of Epoxy resin embedding e.g. the possibility of cutting undecalcified hard tissues and sections for serial reconstruction.     

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.     

Spontaneous Autoradiographic Grain Activation Associated with Mast Cells in Methacrylate Plastic Embedded Tissue Sections

Autoradiographic tracing using tritium labeled compounds or cells is a common laboratory technique for light and electron microscopy. This report describes a chemo-graphic effect associated with certain cells in sections from tissues embedded in the new methacrylate plastic embedding compounds. When tissue sections from rats and rhesus monkeys that received no radioisotope were coated with nuclear track emulsion and subsequently developed, cells with morphologic characteristics of mast cells showed significant grain formation over the entire cell. Three different types of methacrylate plastics were tested using rat and monkey tissues and all three were found to promote grain formation over mast cells; however, this phenomenon was not seen in similar tissue sections from paraffin or epoxy embedded material. The properties of methacrylate plastics which promote positive chemography by mast cells may reflect the greater permeability of this class of plastics. Due to their wide tissue distribution, the presence of such chemographically active cells could cause false estimates of the distribution of either exogenous radiolabeled cells or radioisotopes within many tissues.     

Spontaneous autoradiographic grain activation associated with mast cells in methacrylate plastic embedded tissue sections

Autoradiographic tracing using tritium labeled compounds or cells is a common laboratory technique for light and electron microscopy. This report describes a chemographic effect associated with certain cells in sections from tissues embedded in the new methacrylate plastic embedding compounds. When tissue sections from rats and rhesus monkeys that received no radioisotope were coated with nuclear track emulsion and subsequently developed, cells with morphologic characteristics of mast cells showed significant grain formation over the entire cell. Three different types of methacrylate plastics were tested using rat and monkey tissues and all three were found to promote grain formation over mast cells; however, this phenomenon was not seen in similar tissue sections from paraffin or epoxy embedded material. The properties of methacrylate plastics which promote positive chemography by mast cells may reflect the greater permeability of this class of plastics. Due to their wide tissue distribution, the presence of such chemographically active cells could cause false estimates of the distribution of either exogenous radiolabeled cells or radioisotopes within many tissues.     

DNA polymerase activity in embryos of Allium cepa seed

A low level of DNA polymerase (deoxyribonucleosidetriphosphate: deoxynucleotidyltransferase; E.C. 2.7.7.7) activity can be detected by autoradiography after incubating frozen sections of unimbibed onion seed embryos in an aqueous solution of tritium labelled deoxynucleoside triphosphate. The enzyme is sensitive to sulfhydryl reagents, and incorporation was reduced by high levels of deoxyribonuclease, but inclusion of cycloheximide, ribonuclease, or nucleoside triphosphate did not change activity. On germination, activity remained relatively constant and at a low level until 30 hr after imbibition when it began to increase significantly.     

A Rapid Celloidin Method for the Rotary Microtome

A method is described which combines the writer's hot celloidin technic¹ with a form of the clearing-before-cutting procedure. The method requires only 16-17 days and yields a block which may be cut in any microtome, the sections being as thin as those afforded by paraffin with comparable material. The advantages of celloidin over paraffin, listed in the writer's earlier paper, are retained in the present method which, altho consuming more time than the hot process, requires less skill and gives superior results.     

A Rapid Method for Preparing Paraffin Serial Sections of Teeth and Their Supporting Structures in the Dog

A method of preparing dogs' teeth for serial sectioning involving the following steps is described. The tissues are fixed in 10% formalin followed by partial dehydration in 80% iso-propyl alcohol; decalcified in 5% nitric acid in 10% formalin; washed for 5-8 hours in running water and neutralized in 5% aqueous sodium sulfate for 24 hours, followed by further washing in running water for another 24 hours. They are then dehydrated over a period of 4 days in increasing grades of iso-propyl alcohol and embedded in tissue-mat in sub-atmospheric pressure of 10 lb. Sections so obtained were better suited for cytological study than celloidin preparations, and serial sections could be obtained in a much shorter time.     

A Celloidin Infiltration-Frozen Section Sequence for Enhanced Preservation of Phosphatases in Bone

The effect of the following embedding procedures on the acid and alkaline phosphatase content of decalcified mouse tibiae has been studied: embedding in 23% gelatine for 18 hr at 37° C, embedding in paraffin wax in vacuo for 1 hr at 58° C, and impregnation with 4% celloidin in diethyl ether and ethanol at 4° C for 2-3 days. Unsupported tissues were also used to demonstrate these enzymes for comparison with the above procedures. Tibiae were first fixed in 10% neutral formalin at 4° C for 15 hr, decalcified in equal volumes of 2% formic acid and 20% sodium citrate at pH 4.9 for not more than 5 days and then washed in distilled water before carrying out the embedding schedules. The celloidin-impregnated tibiae were placed in 70% ethanol to harden the celloidin and then washed in distilled water for 1-2 hr. These tibiae and those embedded in gelatine were cast in a gelatine block which was then hardened in 10% neutral formalin at 4° C for 2 hr. Sections of these and unsupported tibiae were cut at 15 μ on a freezing microtome. Decalcified tibiae embedded and blocked in paraffin wax were sectioned at 15 μ on a base sledge microtome. The enzymes were demonstrated using the coupling azo dye method given by Pearse (Histochemistry, 1st Ed. 1954). The stable diazotates of 4 benzoyl amino 2-5 diethoxyanilene, 3 nitro toluidine and o-dianisidine were used. Of the embedding procedures paraffin wax embedding produced the greatest loss of both enzymes. Gelatine embedding and infiltration with celloidin were equally good for the demonstration of acid phosphatase but for alkaline phosphatase the celloidin method was superior. The gelatine embedded material did not produce consistently good results. Celloidin-impregnated tibiae could be stored without marked deterioration of the enzyme content for longer than gelatine-embedded tibiae.     

An Improved Fixing Solution for Methylene Blue Preparations

A method is described which combines the writer's hot celloidin technic¹ with a form of the clearing-before-cutting procedure. The method requires only 16–17 days and yields a block which may be cut in any microtome, the sections being as thin as those afforded by paraffin with comparable material. The advantages of celloidin over paraffin, listed in the writer's earlier paper, are retained in the present method which, altho consuming more time than the hot process, requires less skill and gives superior results.     

Quick Staining Method For Frozen Sections

Since the advent and general acceptance of frozen sections in histological and pathological laboratories it has been necessary to devise methods for staining these sections. The usual method is fixing the tissue to a slide by the use of celloidin. This paper is an attempt to describe a permanent, quick method of staining frozen sections without distortion or mechanical tearing of the tissues.     

Notes on Technic: A Device for Centering Tissue Blocks on the Porter-Blum Microtome

A method of double embedding fixed tissues in 3% low viscosity nitrocellulose and paraffin is described. Five percent phenol in 80% alcohol during dehydration and 5% glycerin in the nitrocellulose solutions enhance cutting qualities. A modified Ruyter's solution is used to flatten sections. After a section is aflixed to a slide, it is passed through chloroform and acetone to remove the paraftin and celloidin. A 1% celloidin dip insures adherence of the seaion to the slide. Slides are stored in 70% alcohol until they are to be stained. Following staining and dehydration in graded alcohols, clearing should be done in a 1: 3 mixture of terpineol and toluene.     

THE INCORPORATION OF TRITIUM FROM THYMIDINE INTO PROTEINS OF THE MOUSE

Tritium from methyl-H³-thymidine was found to be incorporated into proteins in mice. This incorporation in the mouse as a whole represented between 1 and 10% of the injected tritium. Tritiated water was not an intermediate. Transmethylation reactions are proposed as a means whereby certain amino acids might have acquired the tritium from thymidine at some stage of its catabolism. The initial (2 hr) ratios of DNA to protein tritium activities per milligram of wet tissue ranged from 5 in two tissues of low DNA synthetic activity (pancreas, liver) to 35 to 40 in two tissues of high DNA synthetic activity (spleen, small intestine). Labeled nuclear protein was coincident with labeled DNA in nuclei, where it constituted less than 2.5% of the total tritium. The significance of the findings is discussed.     

Double Embedding Again

A method of double embedding fixed tissues in 3% low viscosity nitrocellulose and paraffin is described. Five percent phenol in 80% alcohol during dehydration and 5% glycerin in the nitrocellulose solutions enhance cutting qualities. A modified Ruyter's solution is used to flatten sections. After a section is aflixed to a slide, it is passed through chloroform and acetone to remove the paraftin and celloidin. A 1% celloidin dip insures adherence of the seaion to the slide. Slides are stored in 70% alcohol until they are to be stained. Following staining and dehydration in graded alcohols, clearing should be done in a 1: 3 mixture of terpineol and toluene.     

Metabolism of organically bound tritium in man

The classic methodology for estimating dose to man from environmental tritium assumes that all tritium, whether organically bound or free, enters directly into man's free body water compartment and is uniformly distributed as tritiated water. This methodology ignores the fact that organically bound tritium in foodstuffs may be directly assimilated in the bound compartment of tissues without previous oxidation. A four-compartment model consisting of a free body water compartment, two organic compartments, and a small, rapidly metabolizing compartment is proposed. The utility of this model lies in the ability to input organically bound tritium directly into organic compartments representing tissue solids. The model will be used to illustrate the potential importance of organically bound tritium to cumulative dose estimates. It is found that organically bound tritium in foodstuffs can increase cumulative total body dose by a factor of 1.7-4.5 times the free body water dose alone, depending on the bound-to-loose ratio of tritium in the diet.     

Curdled Milk for Supporting Tissues in Celloidin Embedding

To make a material for supporting tissues during celloidin embedding, milk curd is hardened in ordinary strong alcohol, cut into suitable shapes, dehydrated, extracted with ether, and preserved indefinitely in 1:1 alcohol-ether. The material, which consists of the precipitated proteins of milk, is embedded and sectioned with the tissue. It presents no detectable opposition to the passage of the microtome-knife.