Chromatography and Biological Stains: II. Column Chromatographic Separation of the Colored Materials in Commercial Sudan III

Commercial Sudan III was separated into various fractions by the usual column chromatographic technics using Celite-silicic acid as the adsorbent and commercial petroleum ether (B) as the solvent. By analysis, using both spectrophotometric and paper strip chromatographic methods, it was possible to identify fractions from the commercial sample, including several fractions which were indubitably very highly purified Sudan III. In addition several other fractions containing mixtures of colored components have been isolated.     

Lipid quantitation in formalin-fixed liver sections

We describe a simple, sensitive, and quantitative procedure for measurement of triglycerides and protein contents in formalin-fixed liver sections. The method can detect as little as 0.27 microgram of triglycerides per mg of protein. It is based on selective binding of Sudan IV and Fast Green FCF to fat and total proteins, respectively, and their sequential elution with solvents. Sudan IV is eluted readily with acetone and Fast Green with NaOH-methanol, and the absorbances obtained at 500 and 610 nm can be used to determine the amount of triglycerides and total protein. The color equivalence for Fast Green was obtained after destaining the sections and measuring the protein contents by micro-Kjeldahl analysis. The color equivalence of Sudan IV was estimated by determining the triglyceride content in liver homogenates by an enzymatic procedure and then measuring the amount of dye bound to multiple fixed sections. There was a strong linear correlation between the triglyceride content as determined chemically and that obtained using the equivalence colors (r2 = 0.98). This method is useful to measure fat content in tissue samples and could be applied to evaluate the progression of liver disease.     

Expression of class III beta-tubulin in neuroendocrine tumours of gastrointestinal tract

Class III b-tubulin is presented as a specific marker for the cells of neuronal origin as well as for the tumours originating from these cells. Its expression is considered one of the earliest events that appear in the cells revealing neuronal differentiation. Using monoclonal antibody TU-20 in an immunohistochemical analysis, we studied the expression of class III b-tubulin in gastrointestinal carcinoid tumours. Paraffin-embedded, formalin-fixed tissue sections from 49 tumour samples obtained from following locations: stomach (4 cases), small intestine (8 cases), appendix (18 cases), rectum (3 cases), pancreas (5 cases), liver metastases (7 cases) and lymph node metastases (4 cases) were used in the study. In 41 of the 49 tumour samples (83.7%), positive staining for class III b-tubulin was detected, while 8 tumour samples (16.3%) were negative. Expression of class III b-tubulin was prominent in all three rectal carcinoids and in three atypical carcinoids located in small intestine. Pancreatic neuroendocrine tumours revealed either weak immunostaining (2 cases), or were negative for this marker (3 cases). The intensity of class III b-tubulin immunolabelling was not related to the degree of tumour differentiation. The results of this study suggest that class III b-tubulin could be a perspective marker for gastrointestinal neuroendocrine tumours. Moreover, the differences in its expression could also elucidate some aspects of histogenetic relationships of neuroendocrine tumours of gastrointestinal tract.     

The Use of Sudan Black B as a Bacterial Fat Stain

Sudan black B was introduced as a specific fat stain for the detection of lipids in tissue sections by L. Lison in 1934. Saturated solutions of Sudan black B in 70% alcohol or in ethylene glycol stain the fat bodies of bacteria a deep blue-black color, and this dye is recommended as superior to the other Sudans.

The method used in staining the bacteria was to suspend a loopful of the cells in a drop of the stain solution and to prepare flat wet mounts. The organisms giving positive fat tests with Sudan black B included Bacillus cereus, Bacillus mycoides, Azotobacter beijerinckii, Rhizobium leguminosarum, Mycobacterium avium, Mycobacterium leprae, Oospora lactis, Bacillus tumescens, water spirilla, and fungi.     

Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in Polyethylene Glycol-Glycerol

Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general.     

An Orcein-Oil Red O Stain for Concomitant Demonstration of Elastic Tissue and Lipid

Orcein, 0.5% in 50% isopropanol, 0.5-1 hr, followed by saturated oil red O in isopropanol diluted 3:2 with distilled water, 10-15 min, was used to demonstrate lipids and elastic tissue simultaneously in 10 μ frozen sections of formalin-fixed aortas of the wild African buffalo, showing atherosclerotic lesions. A comparison was made with the oil red O-aldehyde fuchsin (AF) method of Kwaan and Hopkins (Stain Techn., 39: 123-5, 1964) and the resorcin fuchsin (RF)-oil red O method of Lillie (Histopathologic Technic and Practical Histochemistry, McGraw-Hill, 1954), but both gave marked background staining by AF or RF that obscured the smaller deposits of lipid. Sudan IV could be substituted for oil red but did not demonstrate many of the finest deposits of lipids. Sudan black, in combination with orcein, AF or RF, was very satisfactory for demonstrating lipids but obscured many elastic fibres. Sudan dyes I, II, III, brown, blue, and green, with orcein, AF or RF, showed less contrast between lipids and elastic tissue or failed to stain the lipids adequately.     

The effect of Sudan III on drug metabolizing enzymes

We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol (Sudan III). Marked increases were observed in the levels of cytochrome P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7-ethoxycoumarin O-deethylase (ECD) activities. On the other hand, aminopyrene N-demethylase activity was not significantly increased. Further, induced ECD activity was inhibited 90% by a specific antibody against cytochrome P-448 while the inhibition observed with an antibody against cytochrome P-450 was less than 25%. Simultaneous administration of Sudan III and 3-methylcholanthene (3-MC) induced cytochrome P-448 up to a level brought about by either Sudan III or 3-MC treatment alone. In contrast, Sudan III did not induce cytochrome P-448 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes from Sudan III-treated rats showed an identical sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) pattern with those from 3-MC-treated animals. It is concluded that the cytochrome P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for cytochrome P-448.     

Semiquantitative assessment of c-erbB-2 (HER-2) status in cytology specimens and tissue sections from breast carcinoma

OBJECTIVE: To determine whether various methods of fixation of surgical pathology specimens from breast carcinomas would influence the outcome of evaluation of the expression levels of c-erbB-2 (HER-2). For this, comparisons were made between (1) alcohol-fixed (95%) and air-dried smears from fresh surgical pathology specimens of breast carcinomas, and (2) formalin-fixed, paraffin-embedded tissue sections of the same specimens. STUDY DESIGN: Alcohol-fixed and air-dried smears or touch preparations were made from 30 fresh mastectomy/lumpectomy surgical pathology specimens from breast carcinomas. Immunohistochemistry was performed using the c-erbB-2 primary antibody against the extracellular domain of the c-erbB-2 gene product. Staining was simultaneously performed on formalin-fixed, paraffin-embedded tissue sections of the same specimens. A semiquantitative approach was used for evaluation of immunostaining by three independent investigators, and a consensus was reached. RESULTS: A total of 30 cases were reviewed. Tissue positivity was determined for c-erbB-2 in: 73% of alcohol-fixed specimens (n = 13 [3+] and n = 9 [2+]), 67% of air-dried smears (n = 9 [+3] and n = 11 [+2]) and 47% (n = 8 [+3]) and n = 6 [+2]) of formalin-fixed, paraffin-embedded tissue specimens. All formalin-fixed tissue specimens that were determined to positively express c-erbB-2 were also found to be positive on the alcohol-fixed smears. CONCLUSION: The incidence of c-erbB-2 expression in fresh cytologic material is significantly higher (P < .05) than in formalin-fixed, paraffin-embedded tissue. Alcohol-fixed smears demonstrate a slightly higher percentage of cell staining and stronger intensity of c-erbB-2 expression than the matched, air-dried smears. This is a sensitive and simple processing method that can be routinely applied in surgical pathology or fine needle aspiration biopsy specimens for the detection of c-erbB-2 (HER-2), with clinical implications.     

Reduction of Brain Antioxidant Defense Upon Treatment with Butylated Hydroxyanisole (BHA) and Sudan III in Syrian Golden Hamster

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1. 4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.Deceased 4th November 1998     

Immunohistochemical localization of the immunodominant differentiation antigen lacto-N-fucopentaose III in normal adult and fetal tissues

Monoclonal antibodies were produced by immunizing rats with human small cell lung carcinoma (SCLC) cell lines. Monoclonal antibodies 600D11 and 624A12 were found to be directed against the ceramide pentasaccharide that contains the lacto-N-fucopentaose III (LNFP III) sequence of sugars, an isomer of the Lewis A blood group antigen. LNFP III is an immunodominant antigen whose reactivity is maintained in formalin-fixed paraffin-embedded sections (PS). LNFP III has been recognized in a number of human tumors including: SCLC; adenocarcinomas of the breast, gastrointestinal tract, genitourinary tract, and lung; renal cell carcinoma; neuroblastoma; and myelogenous leukemia. We now report the normal adult and fetal tissue distribution of the LNFP III antigen by immunoperoxidase staining on PS utilizing 600D11 and 624A12. Binding was demonstrated in bronchial epithelium and bronchial glands; squamous epithelium of the esophagus; gastric crypts, duodenal enterocytes and Brunners glands; argentaffin cells; jejunal and colonic goblet cells; pancreatic acinar cells; salivary glands; endocervical and exocervical cells; skin epidermis; myelinated motor fibers; cells of the adrenal medulla and anterior pituitary gland; polymorphonuclear leukocytes (PMNs); tissue macrophages and renal proximal tubules and loops of Henle. Staining was localized to cell membranes and within the cytoplasm, with greatest intensity at the apical and basal portions of the cells. These staining patterns were noted in adult and neonatal tissues, and initial expression could be traced to approximately the second trimester of fetal development. Knowledge of the normal tissue distribution of this immunodominant antigenic determinant may offer insight into its structural and functional role in benign and malignant tissues.     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

Cryofixed, freeze-dried and paraffin-embedded skin enables successful immunohistochemical staining of skin basement membrane antigens.

Conventional chemical fixation and paraffin-embedding procedures give good preservation of morphology, although the antigenicity of many proteins in the tissue sample is destroyed. On the other hand, fresh frozen sections can preserve the antigenicity, but provide poor morphological preservation. To overcome this dilemma, cryofixation and freeze drying were used on human skin tissue, applying methodology which has only been used to study lymphoid tissue. First, fresh human skin was cryofixed in liquid isopentane (-160 degrees C) cooled by liquid nitrogen. The skin was then freeze-dried at -40 degrees C and 10(-2) atmospheric pressure for 72 h, followed by embedding in paraffin. Sections 4 microns thick taken from this cryofixed, freeze-dried, and paraffin-embedded skin were stained with hematoxylin-eosin or used for immunolabeling with antibodies against basement membrane antigen, including type IV and type VII collagen, bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and GB3 antigen. The morphological preservation of these sections was as good as that of routine formalin-fixed and paraffin-embedded skin sections. The basement membrane was clearly immunostained with all antibodies used, and the intensity of the reaction was as strong as that seen in frozen sections. Evaluation of antigen distribution in conjunction with the detailed skin structure was therefore possible in the same sections.     

Chromatography And Biological Stains V. Isolation And Spectral Analysis Of The Orange Fat-Staining Component Of Sudan Iii

A sample of the orange fat-staining compound present in commercial Sudan III was isolated and purified by chromatographic methods. This material showed the presence of no other contaminating colored compounds when analyzed by paper chromatographic methods. Spectrographic analysis in the visible and ultra-violet ranges shows a strong absorption maximum at 481 mμ, a shoulder at approximately 425 mμ, and a weak absorption maximum at 315 mμ.     

Histochemical Probing of Potato Periderm with Neutral Red: A Sensitive Cytofluorochrome for the Hydrophobic Domain of Suberin

A technique is described which uses the lipid fluorochrome neutral red as a cytochemical probe to detect the hydrophobic domain of the lignosuberin matrix in native and wound periderm of potato tuber. Toluidine blue O is used as a counterstain to quench autofluorescence. The neutral red technique appears to be specific for the hydrophobic/lipid domain of suberin and is significantly more sensitive than Sudan III and IV. The fluorochrome was extensively used on paraffin-embedded tissue with excellent results but also worked on freehand sections of fresh periderm tissue. In tuber tissue undergoing wound-healing, the pattern of suberin fluorescence obtained with the neutral red probe was identical in specificity to the color pattern obtained with Sudan III/IV, but somewhat different than that observed when berberine was used. Results obtained with the neutral red probe and berberine probe visually demonstrated that during ligno-suberin biosynthesis, the depositions of hydrophobic/lipid and phenolic/lignin-like components in potato tuber periderm were separate processes. The deposition of these components does not necessarily require their simultaneous presence because the fluorescence from these probes showed that the components were not consistently present together on the cell walls.     

Optimization of immunohistochemical techniques to detect extracellular matrix proteins in fixed skin specimens

Complete antigen visualization in the context of well-preserved tissue architecture is the goal of all immunohistochemical techniques. Frozen tissue section techniques achieve optimal antigen visualization but preserve tissue architecture poorly. On the other hand, formalin-fixed tissue section techniques preserve tissue architecture very well but result in antigen masking. Enzymatic digestion or salt extraction of formalin-fixed sections has been used to reestablish antigen expression. Recently acid-alcohol-fixed tissue has been used as a successful compromise between tissue architecture preservation and the visualization of cytoskeletal antigens. In an attempt to find an improved immunohistochemical process for non-cytoskeletal antigens, we compared avidin-biotin immunofluorescence staining in frozen, formalin-fixed, and acid-alcohol-fixed tissues. The fixed tissues were either untreated or treated with enzyme digestion or salt extraction. For this study, we examined healing cutaneous wounds in Yorkshire pigs with antibodies to fibronectin, laminin, von Willebrand factor VIII, and keratin. Although tissue architecture was poor, frozen sections provided the best antigen visualization and were therefore used as the standard for complete antigen expression. Formalin-fixed tissues had excellent tissue architecture, but most antigens were completely masked. Pre-treatment technique only partially overcame the antigen masking caused by formalin. In contrast, acid-alcohol fixation preserved tissue architecture almost as well as formalin and sometimes allowed complete antigen visualization; however, laminin and fibronectin were partially masked. Total recovery of the expression of these antigens could be obtained by pre-treating the acid-alcohol-fixed tissue with either hyaluronidase or 1 M NaCl. Therefore, acid-alcohol-fixed tissue appears best for extracellular matrix (ECM) protein immunostaining as well as for cytoskeletal staining. However, certain ECM antigens require hyaluronidase or 1 M NaCl treatment for optimal visualization.     

Sudan Black B And Acetocarmine as a Combination Stain

Epidermis stripped from either fresh or fixed plant organs, or sections of paraffin-embedded or fresh material are placed on a slide and covered with a drop or two of iron-acetocarmine. The stain is intensified by warming the slide over a flame. After a few minutes a drop or two of a saturated solution of Sudan black B in 45% acetic acid is added and a cover slip applied. The preparations cannot be made permanent, but last a few weeks if sealed with a compound such as gum mastic-paraffin, or if the combined stain is drained off and a drop of Karo syrup is added before the cover slip is applied. The acetocarmine produces its usual staining effects, i.e., nuclei dark red and some components of the cytoplasm of certain cells a less intense red. The Sudan black B colors lipid structures an intense blue.     

青少年食蟹猴糖尿病模型的肝脏病理生理学研究

目的通过对胰岛素用量不足条件下链脲佐菌素诱导的青少年食蟹猴1型糖尿病模型肝脏病理生理学的研究,探讨长期高血糖所致青少年食蟹猴肝损伤特点及机制。方法通过静脉注射68 mg/kg的链脲佐菌素,诱导4只3岁的食蟹猴成为1型糖尿病模型,然后经长期的血糖监测和静脉糖耐量实验来评价该模型的可靠性及稳定性,造模4年后,对模型猴进行血生化、PAS染色、苏丹III染色及普通病理和超微病理等指标的检测,另外选取4只健康与模型猴年龄匹配的猴作为正常对照组,同时进行相应的检测。结果与正常对照组比较,糖尿病猴血清学检测指标中总胆汁酸、尿素氮、谷丙转氨酶、谷草转氨酶、胆碱酯酶、乳酸脱氢酶、总胆固醇、甘油三酯和低密度脂蛋白胆固醇明显升高。组织化学染色结果显示,与正常猴比较,糖尿病猴中央静脉区肝实质细胞肿胀,肝细胞PAS染色（糖原染色）加深,苏丹Ⅲ染色（脂肪染色）阳性细胞增多;电镜结果显示糖尿病猴肝细胞内胞质糖原颗粒增多;线粒体电子密度显著增高,结构不清;窦周隙内含有大量脂滴的肝星状细胞明显增多。结论在长期胰岛素用量不足血糖控制不理想的条件下,青少年食蟹猴1型糖尿病模型肝脏特异性的病理改变是肝糖原贮积和含有大量脂滴的肝星状细胞增生,这些病理改变与非酒精性脂肪肝病的病变特点存在显著不同,但其机制目前尚不清楚。     

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.