Zinc Chromate Modification of the Golgi Technic

A modification of the Golgi technic is described in which the reaction takes place in well fixed formalin material. Thin slices (whole sections of adult monkey, cat and rat cerebrum) 2 to 3 mm. thick, from brains fixed 3 to 4 months in 10% formalin, are chromated for two days in 3 g. of zinc chromate dissolved in 98 ml. of distilled water and 2 ml. of formic acid. Slices are then removed, blotted dry and immersed, suspended by a thread, in 0.75% silver nitrate solution for two days. Solution should be changed after the first day. After silvering, the slices are dehydrated rapidly (total time about one hour) in 95% and absolute alcohol, placed in xylene 10 minutes, in low melting point paraffin 10 minutes and embedded in low melting point paraffin. Only surface infiltration is necessary since sections are cut 90 to 100 u. Sections are collected in 95% alcohol, dehydrated in absolute alcohol, cleared in several changes of xylene and mounted in Fisher's Permount. Results with fetal and new born material were not good.     

Rapid Chromosome and Sex-Chromatin Staining with Pinacyanol

This simple and reliable 10-min procedure for producing uniformly and intensely stained, as well as fade-resistant, chromosome and sex-chromatin preparations uses pinacyanol chloride as the dye. Slides are extracted in 5 N HC1 at 20-23 C for 2 min, washed in running tap water for 2 min, stained in 0.25% pinacyanol chloride solution (made up in 70% methanol) for 45 sec, differentiated in Wright's buffer solution (pH 6.4-6.5) for 45 sec, washed in running tap water for 5 sec, dehydrated in 2 changes, 1 min each, of absolute tertiary butanol, cleared in 3 changes of xylene, a minimum of 30 sec each, and mounted in a neutral synthetic resin such as Permount.     

A Chlorate-Formaldehyde Modification of the Golgi Method

Pieces of fresh nervous tissue 3-5 mm thick are put into a mixture of: 6% K₂Cr₂O₇, 40 ml; 5% KClO₃, 20 ml; 20% chloral hydrate, 30 ml; and concentrated formalin (38% HCHO), 10 ml; allowed to fix 3 days, with a daily change of fluid; transferred to 3% K₂Cr₂O₇ for 3 days, with twice daily changes; then to 1% AgNO₃ for 3 days at 20-25° C. Frozen sections are cut, dehydrated, cleared and mounted in Permount with a cover glass. The method gives good results for microglia and oligodendroglia in addition to the usual staining of nerve cells and their processes.     

Alcoholic Thionin Counterstaining Following Golgi Dichromate-Silver Impregnation

Brains of cats that had been fixed 2 months or longer in 10% formalin were cut into 3-6 mm. slices and impregnated by Golgi's dichromate-silver procedure (6% dichromate solution, 4-6 days; 1.5% silver nitrate solution 2 days). Sections 100 µ thick were cut after embedding in low melting point paraffin. Three changes of xylene and three of absolute alcohol were followed by staining 3-5 minutes in a saturated solution of thionin in absolute alcohol. The sections were dipped quickly in absolute alcohol and cleared in xylene, then differentiation was effected by an equal-parts mixture of absolute alcohol and xylene. A final clearing in three changes of xylene and mounting in Permount completed the process. Counter-staining was most successful when applied to freshly cut sections.     

A Safranin-Fast Green Stain for the Differentiation of the Nuclei of Rumen Protozoa

Mounted, deparaffinized sections of rumen ciliates were hydrolyzed in 1 N HCl for 5 min at 60 C and washed in several changes of distilled water. They were then stained in a mixture of equal volumes of 0.1% aqueous solutions of safranin O and fast green FCF. The sections were washed in 3 changes of distilled water for 2 min each, blotted, dehydrated in 2 changes of absolute alcohol of 1 min each, and mounted from xylene. Several fixatives were employed but only Zenker's gave consistent results. The micronuclei showed a densely stained basophilic “core” surrounded by a peripheral zone of acidophilia, whereas the macronuclei were completely basophilic. Similar results were obtained when RNA was extracted with cold perchloric acid. In conjunction with deoxyribonuclease treatment, the Feulgen reaction indicated that the DNA of the micronucleus is concentrated in the basophilic core while the macronucleus shows a uniform distribution of its chromatin. The safranin-fast green procedure has been used for the structural characterization of rumen protozoa and in studies concerning changes in their nuclear morphology.     

India ink staining of proteins on nitrocellulose paper

India ink staining of proteins that have been electrotransfer blotted onto nitrocellulose paper is described. This stain proved to be a useful adjunct to the enzyme-linked immunoelectrotransfer blot technique. It is more sensitive than Coomassie blue, amido black, and fast green stains and is simple to use.     

False Cellular Localization of Aminopeptidase Caused by Resinous Mounting Media

Fresh frozen sections of rat submandibular gland were processed for leucine aminopeptidase localization with L-lencyl-β-naphthylamide hydrochloride and L-leucyl-β-naphthylamide. When the first substrate was utilized stained sections were dehydrated in an ethyl alcohol series, cleared in xylene, and mounted in water-insoluble (resinous) media. Sections were also removed at each stage of dehydration and cleared and mounted with glychrogel. When the second substrate was used, tissues were partially decolorized in 40% ethyl alcohol and mounted directly in glychrogel. Comparison of all sections mounted in glychrogel indicated that there was no variation in cellular localization, regardless of substrate used or degree of dehydration. Nuclei were unstained. After mounting in balsam or synthetic resin the nuclei exhibited an intense stain and the parenchymal reaction was stronger, but diffuse. Progressive staining of the nuclei was observed, microscopically, immediately after applying the resinous mounting medium. The use of an aqueous mounting medium appears to be mandatory in this procedure—glychrogel is recommended.     

An Analysis of Methods for Permanently Mounting Ovules Cleared in Four-and-a-Half Type Clearing Fluids

Ovules cleared in benzyl benzoate-4 1/2 clearing fluid can be permanently mounted in Piccolyte or Permount by replacing the clearing fluid with absolute ethanol, upgrading the ovules in mixtures of ethanol and xylene (3:1, 2:2, 1:3, and xylene), and mounting them in either mountant under the supported coverglass of a Raj slide. Optical sagittal sections through the ovules resemble microtome sections in that the protoplasts are slightly shrunken away from the cell walls. The artifact is common in permanently mounted sections; fixation and paraffin infiltration are usually cited as the causes—its appearance in the whole-mounted ovules is caused by xylene. Although miscible with the clearing fluid, Euparal is the least satisfactory of the standard mountants for permanent preparations of cleared ovules and is best used with an equal quantity of clearing fluid for semipermanent preparations. A large quantity of Euparal in the mountant produces pronounced shrinkage. A method for permanently mounting cleared ovules with the clearing image unaltered employs a mountant which contains the ingredients of Spurr low viscosity embedding medium. Vinylcyclohexene dioxide (10 drops) is combined with diglycidyl ether of polypropylglycol (6 drops) and nonenyl succinic anhydride (26 drops). Ovules treated for 24 hr in benzyl benzoate-4 1/2 clearing fluid are passed through a graded series of clearing fluid-epoxy medium mixtures (3:1, 2:2, 1:3, and pure epoxy medium) at intervals of 15 minutes. One drop of dimethylaminoethanol, the cure accelerator, is then added to the epoxy medium and the ovules are mounted and covered immediately on a Raj slide. The preparation is cured in an oven at 60 C for 24 hr and observed with phase contrast or Nomarski interference optics.     

An analysis of methods for permanently mounting ovules cleared in four-and-a-half type clearing fluids

Ovules cleared in benzyl benzoate-4 1/2 clearing fluid can be permanently mounted in Piccolyte or Permount by replacing the cleaning fluid with absolute ethanol, upgrading the ovules in mixtures of ethanol and xylene (3:1, 2:2, 1:3, and xylene), and mounting them in either mountant under the supported coverglass of a Raj slide. Optical saggittal sections through the ovules resemble microtome sections in that the protoplasts are slightly shrunken away from the cell walls. The artifact is common in permanently mounted sections; fixation and paraffin infiltration are usually cited as the causes--its appearance in the whole-mounted ovules is caused by xylene. Although miscible with the clearing fluid, Euparal is the least satisfactory of the standard mountants for permanent preparations of cleared ovules and is best used with an equal quantity of clearing fluid for semipermanent preparations. A large quantity of Euparal in the mountant produces pronounced shrinkage. A method for permanently mounting cleared ovules with the clearing image unaltered employs a mountant which contains the ingredients of Spurr low viscosity embedding medium. Vinylcyclohexene dioxide (10 drops) is combined with diglycidyl ether of polypropylglycol (6 drops) and nonenyl succinic anhydride (26 drops). Ovules treated for 24 hr in benzyl benzoate-4 1/2 clearing fluid are passed through a graded series of clearing fluid-epoxy medium mixtures (3:1, 2:2, 1:3, and pure epoxy medium) at intervals of 14 minutes. One drop of dimethylaminoethanol, the cure accelerator, is then added to the epoxy medium and the ovules are mounted and covered immediately on a Raj slide. The preparation is cured in an oven at 60 C for 24 hr and observed with phase contrast or Nomarski interference optics.     

Golgi Impregnation After Formalin Fixation

Formalin fixed (10% aqueous) brain from cat, rabbit and man cut to blocks 3-4 mm. thick was placed in a mixture of potassium bichromate, 5 g.; chloral hydrate, 3 g. and water 90 ml. for 24 hours. The specimens were rinsed through 3 changes of water, and transferred through 3 changes of 1% silver nitrate, 1-3 minutes each, then placed for 24 hours in 1.5% silver nitrate. Frozen sections, 40-50 μ were dehydrated and mounted with a cover glass, using Permount. No deterioration of the stain was seen after 5 months. Some brains had been in formalin for 9 months; others only 7 days.     

A modification of the tannic acid-phosphomolybdic acid-dye stain for demonstrating myoepithelial cells in formalin fixed tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin sections are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:1 methanol:acetic acid, rinsed in 9:1 methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

A Modification of the Tannic Acid Phosphomolybdic Acid-Dye Stain for Demonstrating Myoepithelial Cells in Formalin Fixed Tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin section are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:l methanol:acetic acid, rinsed in 9:l methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

Staining Cells on a Membrane Filter with Giemsa Stock and Tap Water

Cells grown on type AM 6 Alpha Metricel Gelman filter membranes are fixed 1-2 min with OsO₄ vapor, washed in distilled water, dehydrated 4 min in 95% and 4 min in absolute ethanol. They are then stained for 2 min in a 1:1 dilution of Giemsa stock solution and an approximately neutral, low mineral content tap water. The stain is removed from the filter by 1 min in 50%, 1 min in 95%, 1 min in absolute ethanol and 1 min in a mixture of absolute ethanol and xylene 1:1. The filter is finally cleared in xylene and mounted with a synthetic resin. This procedure gave polychrome staining on neonatal thymic cells from C57 black pedigreed mice and on human leucocytes, with little or no stain retained by the filter membrane.     

An Eosin-Fast Green-Naphthol Yellow Mixture for Differential Staining of Cytologic Components in Mammalian Permatozoa

Air-dried smears of saline suspensions of mammalian spermatozoa were stained for 10-60 min at room temperature in a mixture of eosin Y, fast green FCF, and naphthol yellow S (0.1% w/v, each dye) in 1.0% aqueous acetic acid. They were then blotted, rinsed in 1.0% acetic acid until no more dye was removed (0.5-1.5 min), blotted and allowed to dry completely, rinsed in xylene and mounted in synthetic resin. In all species examined except the rat, acrosomes were stained greenish blue to bluish green or blue depending on their thickness; in the rat, they displayed more affinity for eosin and were reddish. In all species, spermatozoan nuclei were strongly stained by naphthol yellow. In intact sperm heads, postnuclear cap had a yellowish green appearance. Midpieces of rodent spermatozoa, especially those of younger gametes, were stained bright red while those of ejaculated bull and rabbit spermatozoa were stained blue-green. Cytoplasmic droplets associated with rodent spermatozoa were consistently stained a dark green. In all species, the remainder of the flagellum generally was stained bluish to blue-black. Deductions concerning the morphology of spermatozoa derived from the staining experiments were verified by means of scanning electron microscopy. Because it provides reliable information concerning the morphology of the various components of mammalian spermatozoa, this simple staining procedure should prove applicable to a wide variety of studies involving the morphology of intact spermatozoa     

A New Methylene Blue Technic for Permanent Preparations

1. Tissues stained intra vitam with methylene blue are fixed in a 10% ammonium molybdate solution in physiological saline (or sea water if the tissue is from a marine animal). Fixation time is kept to a minimum. Washing also is reduced to a minimum.

2. Excess fluids are removed from tissues by blotting with a paper or cloth towel before they are put into the succeeding solution. Tissues are taken from the wash water, blotted and placed in a mixture of equal parts of absolute ethyl alcohol and n-butyl alcohol for 30 minutes. They are then blotted and transferred to n-butyl alcohol for 30 minutes. After blotting they are placed in a mixture of one part methyl salicylate and four parts xylene until cleared. Tissues may be mounted whole or prepared for sectioning by embedding in paraffin in the usual way.

3. Tissues fixed, washed, dehydrated and cleared as described retain nearly all of the stain; the time required is greatly reduced; there is no need to chill the dehydrating solutions; cell distortion is much reduced.     

Experimental Cyanine Red: A New Stain for Nucleic Acids and Acid Mucopolysaccharides

A new cationic dye, experimental cyanine red (du Pont), with an absorption maximum of 536 mμ and a pH of 2.9 in 0.5% aqueous solution, is shown to be suitable for staining nucleic acids and tissue materials presumed to contain acid mucopolysaccharides. Mammalian tissues fixed in 10% neutral buffered formalin or Bouin's fluid are dehydrated, embedded in paraffin, sectioned, mounted, deparaffinized, passed through ethanols to water, and stained for 3-30 min in 0.5% experimental cyanine red in water. Differentiation and dehydration in 3 changes (about 1 min each) of n-butanol is followed by clearing in xylene and mounting in resin.     

A novel xylene substitute for histotechnology and histochemistry

Abstract

Propylene glycol methyl ether (PGME) exhibits excellent solvent and coupling properties. A toxicity database provided evidence suggesting that PGME might be a useful substitute for xylene in histotechnology and histochemistry applications. Tissue specimens were fixed, cleared in either PGME or xylene, embedded in paraffin wax, then dewaxed in either PGME or xylene. Sections were treated with the following stains: hematoxylin & eosin (H & E), three special stains of the Gordon/Sweet silver staining method, PAS, and Masson's trichrome, and immunostains including actin, CD3, CD34, CK, CK7/CK9, Ki-67, and ER/PR. The sections were mounted in a resinous medium consisting of PGME and pinene copolymer, then examined under a microscope. Variables such as water tolerance, dimension change, organic solvency, and anti-fading efficacy also were assessed. Depending on the application, PGME performance was equal to or exceeded that of xylene. PGME provided better optical clarity and nuclear detail, did not harden the tissue samples, conserved tissue antigenicity, and was amenable to resinous mounting. Tissues not dehydrated with absolute ethanol also were processed properly. Tissues treated with PGME did not warp or contract compared to those treated with xylene (p < 0.0001). PGME, however, exhibited less organic solvency than xylene. There was no discernible change in the colors of stains in sections processed with PGME even after storage for two years. These results suggest that PGME is a novel xylene substitute for applications in histotechnology and histochemistry.     

Histochemical Demonstration of Carbonic Anhydrase Activity

Fresh tissue slices fixed in chilled acetone for 1 hour and washed in distilled water for 10-30 minutes were incubated for 30-45 minutes at 37°C. in the freshly prepared incubating mixture: filtrate of a mixture of 8% sodium bicarbonate, 100 ml., and MnCl₂·4H₂O, 1 g. After washing in distilled water for 1 hour, they were dehydrated and embedded in paraffin. Sections were cut 15-20μU, deparaffinized, rinsed in absolute alcohol and placed in a 0.1% solution of potassium periodate for 48 hours at 37°C. The mounted sections were counterstained (if desired), dehydrated in alcohol, cleared in xylene (not carbol-xylene) and mounted in balsam. Many brown granules were produced on the sites of enzyme activity by this procedure. The results obtained seem to be in good agreement with previous findings by biochemical determinations.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30-90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30–90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.