Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

Staining of Mitochondria with Aniline-Acid Fuchsin After Zenker-Formol Fixation

It was observed that mitochondria are well-demonstrated by aniline-acid fuchsin staining after Zenker-formol fixation if the sections are not de-Zenkerized. Tests showed that after mordanting in HgCl₂, K₂Cr₂O₇, FeCl₃, or FeSO₄, mitochondria in sections from tissues fixed in neutral buffered formalin could be stained fairly intensely by the same method. Salts of Ag, Ba, Ca, Cd, Co, Cu, Mg, Mn, and Zn were ineffective. If the presence of occasional mercury crystals in the sections is not objectionable, demonstration of mitochondria in Zenkerformol fixed tissues offer speed and additional flexibility in the subsequent use of the blocks as advantages over usual methods.     

A Hematoxylin Staining Mountant for Microorganisms

A gum arabic mounting medium containing iron-hemotoxylin is described. It is suggested for use as a one step process for making permanent slides of fungi and algae where a simple procedure is of importance in routine use, and where structures are being studied that may be damaged, or displaced, by transfer from one solution to another.     

A Stable, High-Contrast Mordant for Hematoxylin Staining

Small, quantities of sulfuric acid will stabilize iron mordants, used in hematoxylin staining, by preserving these solutions against oxidation. The presence of acetic acid in the mordant improves the specificity of the stain. A stable, high-contrast mordant is obtained when both acids are combined with ferric-ammonium sulfate. This mordant, used in combination with fresh alkaline solutions of hematoxylin, has been found especially effective in the staining of certain nuclear and cytoplasmic components of plant cells.     

A Hematoxylin Staining Procedure for Maize Pollen Grain Chromosomes

Studies of postmeiotic chromosome behavior have been impeded by the thick exine and abundant starch grains of maize pollen. Staining pollen grain chromosomes with acetocarmine is tedious and gives inconsistent, often unsatisfactory results. A hematoxylin stain, used in conjunction with the clearing agent chloral hydrate, has been successfully used by the authors to stain chromosomes, nuclei and sperm cells of the maize pollen grain. An ethanol-formaldehyde fixing fluid is used to fix and preserve the pollen samples. The procedure, which is rapid and simple, gives excellent preparations with both fresh and fixed material. Stained preparations do not get darker with time, as is typical of other hematoxylin stained materials.     

Marchi's Staining Method: II. Fixation

Following experimental lesions, spinal cords of cats and rabbits were fixed with acid, neutral, and alkaline solutions. Staining was limited to a chromate-osmic (Marchi's) solution and a chlorate-osmic solution. The following conclusions were drawn:

The presence of an acid in the fixative caused normal myelin sheaths to stain. This effect was reduced by washing tissues before staining, by adding acetic acid to the stain, or by employing a non-formalin fixative. It was, however, at no time entirely obviated.

A study was made of the granular deposits which occur in nearly all Marchi preparations and which are especially confusing if very light backgrounds are obtained.

The staining reactions of the granular deposits were very similar to those of degenerating myelin but some suppression of the granules was obtained by adding KCIO₃ to the formalin fixative.     

Staining Neural End Feet and Mitochondria After Postchroming and Carbowax Embedding

Spinal cord of cat and rabbit was fixed by perfusion with 10% formalin in physiological salt solution followed by a 2-day immersion in 10% aqueous formalin. Further treatment (postchroming) consisted of a 5-day immersion in: K₂Cr₂O₇, 5 gm; CrFl₃-4H₂O, 2 gm; distilled water, 100 ml; followed by 5% aqueous K₂Cr₂O₇ at 38–40°C for 2–4 wks. After thorough washing, blocks were embedded by infiltration first with polyethylene glycol 1000 M. E. and then with Nonex 63B (Gemec Chemicals Co., London, E. C. 2), and casting in the Nonex. Sections were stained, either mounted or unmounted, by modifications of the Bielschowsky-Gros method, and mounted sections by Weigert-Pal's hematoxylin or by Silver's Protargol method. All 3 methods gave apparently complete staining of pericellular end feet and showed also an abundance of mitochondria. Cytologic preservation was much better than that seen after the usual procedures for this type of staining. Retention of lipoid material in the sections is considered to be the cause of efficient staining of end feet and mitochondria.     

A Rapid Silver-Free Method for Staining the Myenteric Plexus

A method is presented for the relatively rapid demonstration of the myenteric plexus. Saturated Sudan black B in 70% ethanol followed by 0.01% aqueous buffered thionin were used on intestinal peels (whole-mounts) to stain myelinated and unmyelinated fibers and neuron cell bodies, respectively. In contrast to accepted silver methods, these two kinds of fibers were distinguished clearly; Schwann cell nuclei and nodes of Ranvier were visible. Preparations had the following attributes: relatively low optical density coupled with high visual contrast, freedom from metallic “mirroring,” low background staining of subjacent muscle fibers, and presentation of a polychromatic picture. The entire procedure was under the complete and repeatable control of the operator. Perikaryon and nuclear morphology were clearly demonstrated. The limitations of this method are that it does not provide good visualization of individual unmyelinated neuronal processes and does not permit preparation of permanent slides.     

Improved Nuclear Staining with Mordant Blue 3 as A Hematoxylin Substitute

For progressive staining 1 g mordant blue 3, 0.5 g iron alum and 10 ml hydrochloric acid are combined to make 1 liter with distilled water. Paraffin sections are stained 5 minutes, blued in 03% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely resemble a good hematoxylin and eosin.     

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.     

Novel Fixation of Plant Tissue, Staining through Paraffin with Alcian Blue and Hematoxylin, and Improved Slide Preparation

Onion (Allium cepa) root tips were fixed in a proprietary solution without aldehyde, toxic metals or acetic acid. Fixed specimens were embedded in paraffin, sectioned on a rotary microtome and mounted on detergent-washed slides without adhesive. Slides with ribbon segments affixed were immersed in 0.2% aqueous alcian blue 8GX in screw-capped Coplin jars in a water bath at 50 C for 1 hr. Excess alcian blue was rinsed off under cold running tap water and the slides were immersed in quick-mixed hematoxylin at room temperature for 15 min. Stained slides were deparaffinized, rinsed with isopropanol, air dried, and coverslips were affixed with resin. Thus, the traditional paraffin microtechnique has been modified at all steps from fixation to finishing slides with coverslips.     

Rapid Nuclear Staining Method for Saccharomyces cerevisiae

Mithramycin was used to stain nuclei in mitotically dividing and sporulating yeast.     

Staining Algal Pyrenoids with Carmine After Fixation in an Acidified Hypochlorite Solution

Pyrenoids but not nucleoli are clearly stained with propionocarmine after a special fixation. The pyrenoid is distinguished as the largest dark purple, intracellular body that stains homogeneously. The general procedure is as follows: unicells and filaments are collected by centrifugation and any liquid discarded; cells are fixed 5 rain in a mixture of 10 volumes of 50% ethanol, 10 of concentrated HC1, and 5 of Clorox; washed in 95% ethanol 3 times or more to remove all chlorophyll and fixative; mordanted for 5 min with a suitably adjusted concentration of ferric propionate in 50% propionic acid (reddish orange in color); stained 5 min in 0.5% carmine in 50% propionic acid heated in a boiling water bath. The material is mounted in 50% propionic acid. The results are reproducible when material is processed in centrifuge tubes and when the optimum time in the mordant and stain is determined for a particular species and then repeated accurately. This technique was successful in staining pyrenoids of all of the 8 species tested, representing 6 algal divisions.     

Staining Pineal Parenchyma by A Modified Hortega Method After Paraffin Embedding

Fresh pineal glands are fixed in 10% formalin at room temperature for about 3 days. After washing, dehydrating and clearing they are embedded in paraffin, sectioned and mounted. The tissues are placed in 10% silver nitrate for 24 hours, washed and impregnated in strong silver carbonate. The sections are reduced in 10% formalin, washed and toned in gold chloride, fixed in 5% hyposulfite, counterstained with erythrosin and mounted in Canada balsam. The processes of the pineal parenchymatous cells of the sheep, cow and man have been successfully stained by this method.     

A Simple Method to Determine Hematoxylin Ripening

A simple spectroscopic method is proposed to control the “ripening” of Delafleld's hematoxylin solution during natural “ripening” or fast oxidation by different agents. The hematoxylin solution has ripened sufficiently for use with tissue sections staining if the absorption is 1.2-1.3 at 560 nm. The hematoxylin solution must first be diluted 15-fold using a 5% ammonium alum solution and the absorption must be measured in 0.5 cm cuvettes.     

A New, Rapid, Microwave-Stimulated Method of Staining Melanocytic Lesions

A new and sensitive method of staining melanocyte lesions is described. Tissue sections covered by a solution of colloidal silver nitrate are exposed to microwaves for 45 sec in a domestic oven to produce clean, crisp staining of melanocytes and melanoma cells, often showing long delicate dendritic cell processes. The staining technique does not stain other pigments or argyrophilic tissues and is shown to be more sensitive than the standard Masson-Fontana procedure.     

A Rapid Combined Fixing and Staining Method for Plant Chromosome Counts

A new method for rapidly preparing slides suitable for chromosome counts by the use of a combined fixing and staining solution involves the substitution of anthraquinone for picric acid in Bouin's formula and the addition of alizarin red S with a metallic salt as a mordant. The fixed smears, after being dehydrated to 95% alcohol, are differentiated in 0.5% sulfuric acid in 95% alcohol saturated with picric acid, washed, cleared and mounted in xylol-balsam. Cymene may be used to intensify the stain. Root tips fixed in the above solution may be dehydrated in dioxan, a paraffin solvent; infiltrated, embedded, sectioned and mounted in the usual way. The sections are subsequently differentiated in picro-sulfuric acid alcohol and cymene. An alternative method of differentiation for this stain is also described.