Further Experiments with the Masson Trichrome Modification of Mallory's Connective Tissue Stain

Several dyes, notably ponceau 2R, azofuchsin 3B, nitrazine yellow, and Biebrich scarlet may replace imported “ponceau de xylidin” in the Masson ponceau acid fuchsin mixture. Of these Biebrich scarlet appears to be the best and may be used without acid fuchsin.

A mixture of equal parts of 5% solutions of phosphomolybdic and phosphotungstic acids is much superior to either acid alone and gives adequate mordanting in 1 minute at 22°C.

With the fast green modification, times in plasma and fiber stains can be reduced to 2 minutes each. With anilin blue a 4-minute plasma stain is required. One-minute final differentiation in 1% acetic acid is adequate.

Primary mordanting of formalin material may be accomplished by 5 minutes in saturated aqueous mercuric chloride or 2 minutes in saturated alcoholic picric acid. Three minutes washing in running water is required after these mordants.     

Modified elastic tissue-Masson trichrome stain

A combined elastic tissue-Masson technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeff's iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Modified Elastic Tissue-Masson Trichrome Stain

A combined elastic tissue-Massou technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeffs iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

Differential dichrome staining of tissue culture monolayers: alternate dyes and possible mechanism.

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Differential Dichrome Staining of Tissue Culture Monolayers: Alternate Dyes and Possible Mechanism

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Lineage infidelity in chronic myeloid leukemia. Demonstration and significance of hybridoid leukocytes

Blood smears from 13 cases of chronic myeloid leukemia (CML) were examined for bigranulated (basophil/eosinophil) cells. The cells were stained with May-Grünwald-Giemsa, toluidine blue, Biebrich scarlet, Adams' reaction, and were reacted for KCN-resistant peroxidase in two cases. A sequential stain (toluidine blue/Biebrich scarlet/Adams' reaction) was applied to 50 cells in each case. Hybridoid cells occurred in all cases with varying frequency. Double granulation was not only found in immature, non-segmented cells but often in mature segmented cells. The chimeric cells were difficult to detect with May-Grünwald-Giemsa. Biebrich scarlet and Adams' reaction being superior in this respect to Biebrich scarlet. Some granules that were positive by Adams' reaction did not stain with Biebrich scarlet. This is in sharp contrast to the normal and is, therefore, interpreted as a granule atypicality. Since under normal circumstances, eosinophilic and basophilic granules can be viewed as mutually exclusive markers of the respective granulocytic lineages, the simultaneous occurrence in CML cells of both markers demonstrates lineage infidelity. Until now lineage infidelity has been reported only in immature cells. However, our results show that lineage infidelity also occurs in mature segmented cells. This indicates that the progenitors of these chimeric granulocytes follow false genetic programs producing cells with profound irreversible neoplastic aberrations.     

The Peracetic-Orcein-Halmi Stain: A Stain for Connective Tissues

A selective stain useful for the study of connective tissues is described. The stain demonstrates elastic and oxytalan fibers as well as fibrils in mucous connective tissues previously undescribed. Reticular fibers are not stained. The stain may be used on sections that have been fresh frozen or fixed in formalin or ethanol. Sections are deparaffinized, washed in absolute ethanol, oxidized in peracetic acid 30 min, washed in running water, stained in Taenzer-Unna orcein 15 min, 37°C, differentiated in 70% ethanol, washed in running water, stained in Lillie-Mayer alum hematoxylin 4 min, blued in running water, and counterstained 20 sec in a modified Halmi mixture of 100 ml distilled water, 0.2 gm light green SF, 1.0 gm orange G, 0.5 gm phosphotungstic acid and 1.0 ml glacial acetic acid. Sections are rinsed briefly in 0.2% acetic acid in 95% ethanol, dehydrated and mounted.     

Staining Plant Tissues with Chlorazol Black E and Pianese III-B

The staining schedule was developed for a study of the mycorrhizae of red pine, Pinus resinosa Ait. From 70% alcohol, sections are stained in a saturated solution of chlorazol black E in 70% alcohol, 10-30 min; free dye removed by washing in 95% alcohol; stained 18-24 hr in Pianese III-b; rinsed in 95% alcohol, acidified by the addition of 2 ml of saturated aqueous picric acid per 100 ml, 3-4 changes or until the last change is pale yellow or light green; and rinsing in 95% alcohol to remove the acid. If the acid fuchsin is too intense, a cautious differentiation with 95% alcohol containing 1-3% of a 0.1 N solution of NaOH is made. If too much chlorazol black is removed, the effect can be compensated by overstaining with this dye at the beginning of the process. Sections are dehydrated, cleared, and covered in the usual manner. This stain has applications to plant tissues generally, and is particularly effective for meristematic tissues. It shows details of cytoplasmic structures and gives sharp delineation of primary cell walls.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

Oil Blue Na as A Stain for Rubber in Sectioned or Ground Plant Tissues

Oil blue NA (Calco), a stain which colors rubber bright blue, has been used effectively in studying the distribution of rubber in several plant species. Fresh or fixed sections are cut, bleached with Javelle water or NaOCl solution, treated with 9% KOH in 95% ethanol, washed with several changes of water and finally with 95% ethanol, and stained with 0.05% oil blue NA in 70% ethanol. Sections are rinsed in 50%' ethanol, placed in 40% glycerin, and mounted in glycerin jelly.

For the detection of changes in the distribution and character of rubber in milled or ground tissues, much the same staining procedure is followed. The stained tissues usually are examined and dissected under a stereoscopic microscope, a procedure which permits rubber to be recognized by both its staining reaction and by a more specific property, elastic elongation.

A microscopic technic is presented whereby it is possible to determine approximately the relative proportion of dispersed and coagulated rubber latex in unstained tissues.     

An Easily Controlled Regressive Trichromic Staining Method

Three modifications of Mallory's connective tissue stain are described and some features of the action of picric acid are discussed.

In the first and most critical method the nuclei are stained in an iron hematoxylin and then differentiated in a picric acid solution containing orange G. This not only differentiates the nuclei, but stains all other elements yellow. The section is then washed in running water to remove the yellow color from all tissues except those which are to remain yellow in the final preparation (usually the erythrocytes). The section is next stained in an acid fuchsin mixture and then differentiated until the desired depth and contrast is obtained. Staining in anilin blue follows and this in turn is differentiated to suit. The section is then dehydrated and mounted.

In the second method the nuclei are stained in hemalum (e.g. Harris's) for a short time; the section is then rinsed and immersed in a mixture of picric acid and acid fuchsin and thereafter is differentiated; it is next passed into anilin blue w. s. and then differentiated and mounted as before. This is less critical than method I, but can be applied to large batches of slides at a time.

The third method is a one-solution method. After staining the nuclei in hemalum, the section is immersed in the “Picro-Mallory” solution, differentiated briefly, dehydrated and mounted. This modification, while being the least critical, is most suitable for routine use when the tissues have been fixed in a fluid containing chromate; the other commonly used fixatives, while giving useful results, are not so good.     

The Assessing of Acidophilia with Biebrich Scarlet, Ponceau De Xylidine and Woodstain Scarlet

Horobin and Bennion stated in 1973 that the bonding of Biebrich scarlet at an alkaline pH is hydrophobic as the isomers of Biebrich scarlet (ponceau de xylidine and woodstain scarlet) do not stain at that pH. If correct this would negate the use of Biebrich scarlet as proposed by Spicer and Lillie in 1961 as a measure of acidophilia. We have found all three isomers to stain similarly at an alkaline pH. Goldstein stated in 1963 that Biebrich scarlet saturated with urea stained elastic fibers only faintly or not at all. This we confirmed; we also found that the staining of other acidophilic structures (Paneth cell granules, sperm heads, etc.) was blacked. If only hydrogen bonding and not ionic bonding u blocked in an aqueous dye solution saturated with urea then the exact meaning of alkaline Biebrich scarlet staining is unknown.     

Stain Technology: A Combined Elastic, Fibrin and Collagen Stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified VerhoefPs elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by Hssamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsia, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

A combined elastic, fibrin and collagen stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified Verhoeff's elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by lissamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsin, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

Oxazine Dyes. 3. Simultaneous Nuclear and Cytoplasmic Staining from Single Solutions

Of 84 dyes tested, 26 were found to give a stable solution with celestine blue B dispersions which simultaneously stained nuclei and cytoplasm. The cytoplasmic dye is dissolved in celestine blue B dispersion prepared by the method of Cray et al. (1956). Croceine scarlet (C.I. 286), in the proportion of 0.38 gm to 114 ml of celestine blue B dispersion, gives results strikingly similar to hematoxylin-eosin when used for 2 min on a wide variety of tissues. No differentiation, other than that which occurs during dehydration, is necessary.     

Azophloxine in Two-Dye and Four-Dye Staining Combination

Tissues are fixed in either 10% formalin or Lavdow-sky's mixture. After the tissues are sectioned and mounted, they are stained in hematoxylin, then counterstained for 2 minutes in 0.1% aqueous azophloxine to which 4 drops of acetic acid have been added to each 100 ml. of stain. Sections are then rinsed in 0.2% acetic acid and dehydrated. Azophloxine GA can be used also in a tetrachrome method. Sections are stained in Harris' hematoxylin, washed, and placed in 0.2% acidified aqueous azophloxine for 2 minutes. They are then rinsed in 0.2% acetic acid, stained 1 minute in an aqueous mixture of 4% phosphotungstic acid and 2% orange G solution and rinsed again in 0.2% acetic acid. Finally, they are stained in 0.2% light green for 2 minutes, and differentiated in 0.2% acetic acid for 5 minutes. The advantage in using azophloxine is that it is clear and delicate and when used in a constant concentration, does not overstain if the recommended procedure is followed.     

Displacement.

Displacement is a noncommital term for the reactions that occur when slides previously stained in phloxine or rose Bengal are immersed for varying lengths of time in a solution of another dye in ethyl Cellosolve. In most histotechnic texts Lendrum's (1947) phloxine-tartrazine is given as the stain for acidophilic inclusion bodies. However the lack between the phloxine and tartrazine has been a serious limitation. A number of dyes were tried as possible substitutes for the tartrazine. A rose Bengal-Bismark brown Y procedure was developed which stains similarly to Lendrum's phloxine-tartrazine and which does have the needed contrast. After staining for 10 min in 1% aqueous rose Bengal and rinsing in isopropyl alcohol slides are placed for 20, 30, 40 and 50 min in 0.05% Bismark brown Y in ethyl Cellosolve. In various tissues and structures the rose Bengal is sequentially displaced by the Bismark brown Y. Thus collagen loses the red stain after 30 min while acedophilic structures like sperm heads and Paneth cell granules retain the red stain after 50 min in the displacement solution. The results are strikingly similar to staining with alkaline Biebrich scarlet.     

Azophloxine in Two-Dye and Four-Dye Staining Combination

Tissues are fixed in either 10% formalin or Lavdow-sky's mixture. After the tissues are sectioned and mounted, they are stained in hematoxylin, then counterstained for 2 minutes in 0.1% aqueous azophloxine to which 4 drops of acetic acid have been added to each 100 ml. of stain. Sections are then rinsed in 0.2% acetic acid and dehydrated. Azophloxine GA can be used also in a tetrachrome method. Sections are stained in Harris' hematoxylin, washed, and placed in 0.2% acidified aqueous azophloxine for 2 minutes. They are then rinsed in 0.2% acetic acid, stained 1 minute in an aqueous mixture of 4% phosphotungstic acid and 2% orange G solution and rinsed again in 0.2% acetic acid. Finally, they are stained in 0.2% light green for 2 minutes, and differentiated in 0.2% acetic acid for 5 minutes. The advantage in using azophloxine is that it is clear and delicate and when used in a constant concentration, does not overstain if the recommended procedure is followed.     

Metallic Lakes of the oxazines (Gallamin Blue, Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2-3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3-5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.