A Two Step Stain for Normal and Leukemic Monocytes Using Two Different Dyes Applied in Sequence

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

A Selective Stain for Eosinophils Using Two Oxazine Dyes Applied Sequentially

A methanolic solution of the oxazine textile dye, C. I. basic blue 122, followed by an aqueous alkaline solution of the oxazine dye, C. I. basic blue 141, and a brief rinse in an acetate buffer at pH 3.45 produces intense black staining of eosinophil granules. This staining was selective for eosinophils while other types of peripheral blood leukocytes showed little if any staining under the same conditions. This staining procedure may be useful for detecting eosinophils in samples of blood, bone marrow, or urine when eosinophiluria results from interstitial nephritis.     

Basic Blue 54: a New Colorant for Monocytes

C.I. basic blue 54, a sulfur containing azo textile dye, stained the nucleus and cytoplasm of normal and leukemic monocytes bright red-violet. Essential for the staining reaction was a brief final rinse in a pH 3.6 acetic acid-sodium acetate buffer. Coloration of the type found in monocytes was not observed in other types of mature and immature leukocytes.     

Rapid Identification of T Helper and T Cytotoxic/Suppressor Lymphocytes with an Oxazine Dye

Peripheral blood lymphocytes displayed a plurality of sues and colors when exposed first to a methanolic solution of C.I. basic blue 141, then to an aqueous alkaline solution of the Same dye and Msed in a neutral HEPES buffer containing trace amounts of various salts. As confirmed with purified lymphocyte subpopula-tions obtained with a cell sorter, T helper cells (CD4) were small and their nuclei and cytoplasm stained deep blue. T cytotoxic/suppressor cells (CD 8) were larger than T helper cells, their nuclei stained pale green or blue green and their cytoplasm contained a cluster of magenta colored granules. From start to finish, the stain takes 15 min to perform. Used in the manner described, basic blue 141 holds promise as a rapid means of identifying and differentiating CD4 and CD8 cells under the ordinary light microscope without using monoclonal antibodies or fluorescence.     

New stains for blood and bone marrow cells.

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an asymmetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.     

New Stains for Blood and Bone Marrow Cells

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an assymetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.     

Basic Blue 148: A Rapid Stain for T Helper Cells

After brief exposure to an aqueous solution of the oxazine textile dye C. I. basic blue 148 following fixation in 37% formalin, 95% ethanol and glacial acetic acid, T helper cell nuclei and cytoplasm in specimens of peripheral blood displayed a deep red-violet color. No other cell in normal blood or bone marrow specimens showed intense staining of this type. The total staining time is 1 min. Basic blue 148 stain is a promising technique for hematology and immunology laboratories as a rapid screening test for T helper cells in blood specimens using a microscopic slide and ordinary incandescent illumination.     

Lycramine brilliant blue JL: a new stain for human megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte cytoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

Lycramine Brilliant Blue Jl: A New Stain for Human Megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte ctyoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

Basic Blue 75: a New Stain for Erythroblasts

C.I. basic blue 75 in an aqueous alkaline solution stains the nuclei of mature anc immature erythroblasts bright red. Simultane ously, the stain colors the cytoplasm of erythro blasts blue in immature cells and purple in ma ture cells. Colors of the type described were noi found in other normal and abnormal hemato poietic cells.     

C. I. acid red 52: a new stain for cells of granulocytic origin

Using the xanthene dye C.I. acid red 52 (C.I. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52. In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

Use of eriochrome cyanine R for routine histology and histopathology: an improved dichromatic staining procedure

Abstract

A modified dichromatic iron-eriocyanine R (Fe-ECR) staining method is described. Staining obtained with this new technique generally was similar to that of hematoxylin and eosin (H & E). Cell nuclei were stained blue. Cardiac, smooth and skeletal muscle, and red blood cells, were stained different shades of red. Collagen fibers were stained different shades of orange, usually faintly. Decalcified bony tissue was stained pinkish violet. Epithelial cells were strongly stained deep shades of red, magenta and violet. Cartilage matrix, and goblet and mast cells were unstained. Although Fe-ECR staining differed too much from standard H & E staining to be a substitute for diagnostic purposes, the dichromatic method described might usefully replace van Gieson or trichrome stains, especially if muscle is of interest. A pH 0.95 staining solution was used to differentiate initially over-stained sections followed by washing in distilled water. This dichromatic technique is easier to perform and more precisely controllable than other ECR dichromatic methods. The entire procedure can be completed in less than 5 min. The technique has the advantages of greater technical simplicity and speed, a larger range of polychromasia, and a longer shelf-life than H & E. ECR also is more reliably available than hematoxylin and usually is less expensive.     

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with Sörensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.     

Recovery and Enhancement of Faded Cleared and Double Stained Specimens

Cleared and stained specimens may become faded and/or opaque after long periods in preservative solutions. We developed a technique to revitalize faded and/or destained specimens regardless of their age. By adapting and revising Wassersug's procedure for differential staining of bone and cartilage, we successfully cleared and restained numerous small amphibian specimens of various ages. After rehydrating, specimens were bleached in potassium hydroxide and hydrogen peroxide, stained with alcian blue, and macerated with trypsin as needed. Alizarin red stain was applied or reapplied, although staining occasionally was unsuccessful in old formalin fixed specimens. After restaining, the specimens were dehydrated and placed in glycerol.     

C. I. Acid Red 52: A New Stain for Cells of Granulocytic Origin

Using the xanthene dye C.I. acid red 52 (CI. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52- In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

A new stain for identification of avian leukocytes

Differential staining of avian leukocytes was achieved within 6 min following brief fixation in a methanolic solution of C.I. acid red 360 followed by immersion in a mixture containing C.I. basic blue 41, C.I. basic blue 141, and C.I. acid red 52. Heterophils contained black angular and punctate granules. Eosinophils contained bright purple granules. Lymphocytes displayed red nuclei and blue cytoplasm. Monocytes contained red-brown nuclei and lavender cytoplasm. Basophils showed red-orange granules. Thrombocytes stained deep purple. Compared to traditional panoptic stains like Wright's or Giemsa's, the new staining method provides brighter colors, more precise details of cellular structures, and shorter staining time. Significantly, it facilitates identification of avian leukocyte species based on differences in color as well as differences in size and shape.     

Recovery and Enhancement of Faded Cleared and Double Stained Specimens

Cleared and stained specimens may become faded and/or opaque after long periods in preservative solutions. We developed a technique to revitalize faded and/or destained specimens regardless of their age. By adapting and revising Wassersug's procedure for differential staining of bone and cartilage, we successfully cleared and restained numerous small amphibian specimens of various ages. After rehydrating, specimens were bleached in potassium hydroxide and hydrogen peroxide, stained with alcian blue, and macerated with trypsin as needed. Alizarin red stain was applied or reapplied, although staining occasionally was unsuccessful in old formalin fixed specimens. After restaining, the specimens were dehydrated and placed in glycerol.     

An Automated Double Staining Procedure for Bone and Cartilage

Differential skeletal staining is an important part of developmental toxicologic studies. Traditionally these studies have required time-consuming differentiation of one or both stains used and careful attention to the maceration step to prevent specimen destruction. We present a fully automated protocol which does not require differentiation of either dye and incorporates a controlled maceration step which is highly reproducible. This has resulted in high quality staining that is reproducible, stable, and can be done in volume with minimal personnel time. The process involves the staining of skinned, eviscerated specimens fixed in 95% ethanol. Using an automated tissue processor, the specimen is stained in alcian blue for 24 hr, macerated in 3% potassium hydroxide for 24 hr and stained with murexide for 24 hr. The specimens are cleared and preserved in glycerol. Within three days specimens have red stained bone and blue stained cartilage. The procedure was developed using 20-day-old Sprague-Dawley rat fetuses to evaluate the feasibility of using the procedure for teratology studies involving the fetal skeleton. Evenly stained specimens can be examined within three days and stored for years without loss of staining.     

A new stain for identification of avian leukocytes

Differential staining of avian leukocytes was achieved within 6 min following brief fixation in a methanolic solution of C.I. acid red 360 followed by immersion in a mixture containing C.I. basic blue 41, C.I. basic blue 141, and C.I. acid red 52. Heterophils contained black angular and punctate granules. Eosinophils contained bright purple granules. Lymphocytes displayed red nuclei and blue cytoplasm. Monocytes contained red-brown nuclei and lavender cytoplasm. Basophils showed red-orange granules. Thrombocytes stained deep purple. Compared to traditional panoptic stains like Wright's or Giemsa's, the new staining method provides brighter colors, more precise details of cellular structures, and shorter staining time. Significantly, it facilitates identification of avian leukocyte species based on differences in color as well as differences in size and shape.