A simplified aluminum stain in paraffin sections of bone from hemodialysis patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Histochemical demonstration of aluminum and iron deposition in pulmonary bony tissues in three cases of diffuse pulmonary ossification

Diffuse pulmonary ossification is a rare condition. We examined three cases of it in Japan, and attempted histochemically to stain for deposition of aluminum and iron in bony tissues. The patients were all female, and in their mid-twenties, mid- eighties, and later teen years. One of the patients had been exposed to heavy metals in her work involving heavy-metal analyses for 18 months. Aluminum staining and Berlin blue staining for iron were performed with dewaxed, undecalcified sections of pulmonary tissues from these three cases. Interestingly, all pulmonary bony tissues from the three cases examined exhibited linear regions of both aluminum and iron deposition in the calcifying fronts or the cement lines of bones. The patient exposed to heavy metals exhibited the most severe aluminum and iron deposition, and also exhibited positive reaction for both aluminum and iron in elastic fibers of blood vessels. Foreign body granulomas with multinucleated giant cells exhibiting elastophagia were also found in this case. This phenomenon, "endogenous pneumoconiosis", appeared to have been the cause of pulmonary hemorrhage in this case, resulting in focal heavy hemosiderosis. It is of great interest that identical patterns of aluminum and iron deposition in hemodialysis patients were found in these three cases, This is the first report on histochemical demonstration of aluminum and iron deposition in diffuse pulmonary ossification, and detailed analysis of additional cases is needed.     

A Tri-Basic-Dye Stain for Nerve Cells

A new staining method has been developed for the study of nerve cells and Nissl granules which combines three basic dyes, cresylecht violet, toluidine blue and thionin. The use of this tri-basic-dye stain results in finished preparations that are critically stained and permanent. Paraffin sections (4 μ sections preferably) are mounted on slides by the starch medium, deparaffinized and stained by the tribasic staining solution. After differentiation in acidified distilled water, sections are dehydrated, returned to stain solution and again dehydrated, then cleared and mounted in Clarite. Various vertebrate material including normal and pathological human tissues have been stained with this triple dye solution. Especially for pathological material, re-immersion of slides in the staining and 80% alcohol solutions before mounting, differentially intensifies the staining reaction. Fixatives used were 10% formalin, 95% alcohol, Bouin and formalin-Bouin (10% formalin followed by Bouin).     

Effects of prolonged water washing of tissue samples fixed in formalin on histological staining

The effects of prolonged water washing after fixation for 48 h in 10% (v/v) phosphate-buffered neutral formalin on the quality of representative histological staining methods were evaluated using samples of liver, kidney, spleen and thymus collected from three male Crl:CD(SD)(IGS) rats and one male beagle dog. Because door-to-door courier services in Japan prohibit handling formalin, our goal was to confirm that formalin fixed wet tissue samples could be stored in tap water rather than formalin during transportation of the samples without decreasing the quality of their staining or immunohistochemistry. Each tissue sample was allocated randomly to one of three groups: 12 min, 3 days and 7 days of washing in running tap water; samples then were routinely embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin, periodic acid-Schiff, azan, and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Immunohistochemical staining for Factor VIII, ED-1 and CD3 also was assessed. Prolonged water washing for up to 7 days did not affect the morphology or stainability by standard histological methods, or the intensity and frequency of positive reactions using the TUNEL method. Only immunohistochemical staining of Factor VIII was altered in both the rat and dog sections after 7 days of water washing. The intensity of positive reactions of Factor VIII immunohistochemistry after 7 days water washing was still strong enough to detect microscopically. Therefore, prolonged water washing for up to 7 days after formalin fixation does not have seriously detrimental effects on the quality and characteristics of paraffin sections stained by various methods, including immunohistochemistry.     

Immunofluorescent staining of leptospires in pepsin treated histologic sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

Immunofluorescent Staining of Leptospires in Pepsin Treated Histologic Sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

Iron Gallein Elastic Method—A Substitute for Verhoeff'S Elastic Tissue Stain

A method for staining elastic fibers in formalin fixed, paraffin embedded sections is described. After deparaffinizing and dehydration. sections are stained for 30 minutes in a solution prepared by mixing equal parts of 1% gallein dissolved in ethylene glycol and absolute alcohol (1:4), and 1.16% aqueous ferric chloride in 1% hydrochloric acid. The sections are washed in water and then differentiated in 2% ferric chloride for 2 minutes. After washing in water, the sections am counterstained with a variant of Van Girson's picric acid-acid fuchsin for 1 minute. The results are similar to Verhoeff s elastic stain with elastic fibers staining black. An advantage to this staining procedure is that visually controlled differentiation is not necessary.     

Use of Gallocyanin as a Myelin Stain for Brain and Spinal Cord

Tissue fixed in 10% formalin, formol saline, CaCO₃ or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22-25 C. Mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1-2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na₂CO₃, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5-10 min in a solution consisting of: 0.1 M acetic acid, 60 ml; 0.1 M sodium acetate, 40 ml; methyl green, 500 mg. Washing, dehydration, clearing and mounting completed the process. Myelin sheaths were stained dark violet; neuronal nuclei, light green with dark granules of chromatin; nucleoli of motor cells and erythrocytes, dark violet; cytoplasm, green with dark green Nissl granules. The simple and reliable method can be adapted easily for use with automatic tissue processors.     

Stain for histamine in mast cells in fixed tissue

Summary A technique is described for staining histamine in mast cells of tissues fixed in alcohol. The method employs cold ethanol fixation, xylene clearing, paraffin embedding, and staining of the sections with 1% o-phthalaldehyde in ethyl benzene in a humid chamber. This study was supported by a grant from the John A. Hartford Foundation.     

Advantages in the Use of Aldehyde Fuchsin with Van Gieson's Picro-Fuchsin Counterstaining for Differentiating Bone and Cartilage

Specimens of bone were fixed in 10% neutral phosphate-buffered formalin or in Bouin's fluid and decalcified in 10% formic acid buffered with 10% sodium citrate. Materials were embedded in paraffin and 4-5 μ sections attached to slides were oxidized with 0.5% KMnO₄, decolorized in 1% oxalic acid, stained with aldehyde fuchsin, and counter-stained with Van Gieson's picro-fuchsin. Sections were dehydrated, cleared and mounted in a synthetic resin. Microscopically, the differentiation between bone and cartilage was seen as red and purple respectively, with connective tissue red; muscle and erythrocytes, yellow; and elastic fibres purple. The areas occupied by bone, cartilage and erythrocytes could be compared, and also the depth to which cartilage extended into the ossified sites. The advantages of this staining combination are: good contrasts in colour, ease of applying the stain, and virtual self-differentiation of the staining solutions.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Acceleration of the Movat Pentachrome One Stain by Heat

The conventional staining time for Movat's pentachrome I stain (Arch. Path., 60: 289-295, 1955) was shortened from about 18-19 hr to about 2.5 hr. The ammoniated alcohol and the resorcin-fuchsin staining baths were heated to 56 C. All other steps in the technic were performed at 25-27 C. Staining properties of paraffin sections of many types of tissue fixed in formalin, formol-sublimate-acetic, or in Bouin's fluid, showed that staining with resorcin-fuchsin at the elevated temperature gave the same results as staining at room temperature.     

Niagara Blue 4B As A Fast Simple Stain for the Adenohypophysis

For differentiation of cells of the adenohypophysis, the Niagara blue 4B method requires no special preliminary fixative nor very fresh tissue, and requires no more time than routine hematoxylin-eosin (H-E) staining. The method requires fixation in 10% formalin. After processing to paraffin wax, deparaffinise and hydrate the sections and stain in 1% aqueous Niagara blue 4B solution for 2 min. Stain afterwards with hematoxylin for 1 min then differentiate, wash, dehydrate, clear and mount. This method can be used also for staining old HE slides by removing the covers, applying the Niagara blue 4B and restaining with eosin. The Niagara blue 4B combined with H-E gives the best and most colorful result. This method allows special staining of the adenohypophysis from human post-mortem material to become routine.     

A Triple Stain for Deoxyribonucleic Acid, Polysaccharides and Proteins

Tissues from mammalian, amphibian, insect and plant sources were fixed in formalin or in Carnoy's, Helly's, or Smith's fluid and processed in the usual manner to mounted paraffin sections. These were stained by the sequence: Feulgen reaction (using azure-A-Schiff reagent); periodic acid Schiff (with basic-fuchsin-Schiff reagent); and a 0.02% solution of naphthol yellow S in 1 % acetic acid. Nuclei stained blue to green; polysaccharides, red; and proteins, yellow. Thus, deoxyribonucleic acid, vicinal hydroxy and hydroxy-amino groups, and free basic groups of proteins were demonstrated selectively. Of the tissues tested, stomach, intestine, kidney, thyroid gland and plant root tips were most strikingly stained.     

A bright field/fluorescent stain for aluminum: its specificity, validation, and staining characteristics.

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.     

A bright field/fluorescent stain for aluminum: its specificity,validation, and staining characteristics

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.     

A bright field/fluorescent stain for aluminum: its specificity, validation, and staining characteristics

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.     

Laser Microprobe Mass Analysis (LAMMA) to Verify the Aluminon Staining of Bone

Triammonium aurin tricarboxylate (aluminon) has been used to localize aluminum in 2 μm sections of undecalcified, methyl methacrylate embedded bone obtained from patients with terminal chronic renal failure. Aluminum appeared in four cases as bright red lines at the mineralized-bone boundary. In two cases, however, purplish lines were found and one patient showed red as well as purplish lines. Laser microprobe mass analysis (LAMMA) identified aluminum at the location of the red lines and both aluminum and iron at the purplish lines. Furthermore, both iron and aluminum were found in histiocytic bone marrow cells, which showed brownish aluminon staining. It appears that when aluminum and iron occur together, aluminon staining may yield aberrant results. This study shows that LAMMA can be used for the identification of elements sought by histochemical methods and thus permits the evaluation of their staining effects.     

Laser microprobe mass analysis (LAMMA) to verify the aluminon staining of bone

Triammonium aurin tricarboxylate (aluminon) has been used to localize aluminum in 2 micron sections of undecalcified, methyl methacrylate embedded bone obtained from patients with terminal chronic renal failure. Aluminum appeared in four cases as bright red lines at the mineralized-bone boundary. In two cases, however, purplish lines were found and one patient showed red as well as purplish lines. Laser microprobe mass analysis (LAMMA) identified aluminum at the location of the red lines and both aluminum and iron at the purplish lines. Furthermore, both iron and aluminum were found in histiocytic bone marrow cells, which showed brownish aluminon staining. It appears that when aluminum and iron occur together, aluminon staining may yield aberrant results. This study shows that LAMMA can be used for the identification of elements sought by histochemical methods and thus permits the evaluation of their staining effects.