Automated Feulgen staining with a temperature-controlled staining machine

A Shandon Varistain 24-3 staining machine was modified in order to run automated DNA Feulgen staining. Initial studies showed a strict dependence of the staining intensity (integrated optical density [IOD]) on the temperature of the DNA hydrolysis in 4 N HCl: a difference of 0.5 degrees C around the optimum hydrolysis temperature of 27.5 degrees C resulted in IOD differences of up to 7.8% in epithelial cells and up to 12.0% in lymphocytes. A temperature-controlled stainless steel cuvette, covered with a 4 N HCl-resistant material, was developed and integrated into the machine. Temperature measurements were performed at different positions in the cuvette and on glass slides with copper-constantan electrodes fixed on them; no temperature gradient could be detected within the cuvette. The adjusted temperature of 27.5 degrees C remained constant over 24 hours. The coefficient of variation (CV) of the staining intensity in lymphocytes between different areas on the same slide and between different slides of the same staining cycle was less than 0.6%. The CV between different staining cycles was 5.9%. This system for automated Feulgen staining thus gives reproducible and reliable results and may be introduced into routine diagnostic procedures.     

Understanding Romanowsky staining

Summary Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.     

Understanding Romanowsky staining

Summary Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of toxic granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).To whom offprint should be sent     

Comparison of Papanicolaou staining and Feulgen staining for automated prescreening

Feulgen staining is considered to be a quantitative DNA-specific cytochemical procedure. The applicability of this staining in high-resolution cytometry was tested in comparison with a regressive Papanicolaou staining. Papanicolaou-stained or Feulgen-stained intermediate and carcinoma cells selected by a cytologist were examined with a Zeiss scanning microscope photometer at 546 and 560 nm, respectively. After cell image segmentation and feature extraction, a statistical data evaluation was carried out by computer. Cell distributions with respect to four selected nuclear features demonstrated the influence of the staining procedure on cell feature measurements. The discriminatory power of the classification system as related to both staining procedures was studied using discriminant analysis. Using only nuclear features, a 7.3% improvement of the overall correct classification rate (from 85.0% to 92.3%) was achieved using Feulgen staining. The misclassification rate was simultaneously reduced by 50%. Using cytoplasmic as well as nuclear features, a 98% rate of correct classification was achieved.     

The staining properties of thiazolylazoalkylaminophenol derivatives in histochemical staining of cadmium

In the continuous research for a more sensitive chelator for cadmium, thiazolylazoalkylaminophenol derivatives were synthesized. The staining sensitivity for cadmium and molar absorptivity of their cadmium chelates were examined. Though the molar absorptivity of bromothiazolylazodiethylaminophenol(Br-TADA)-cadmium chelate demonstrated an order of 10(5), the staining sensitivity of Br-TADA was unexpectedly low. The lowering of the sensitivity of Br-TADA may be attributed to alkaline hydrolysis and a decrease in the stability constant of the cadmium chelate.     

免疫组化染色和特殊染色对显示幽门螺杆菌的效果影响对比

目的探讨分析免疫组化染色和特殊染色对显示幽门螺杆菌的效果影响对比。方法选取2013年9月-2014年9月在我院的胃黏膜活检的组织标本共120例,将进行三种染色方法的检测,分为三组。Giemsa组:使用Giemsa染色法进行染色;中红组:使用中性红染色法进行染色;免疫组:使用免疫组织化学法进行染色,比较三组染色的特点。结果结果显示,染色阳性率:Giemsa组为45.0%,中红组为37.5%,免疫组为62.5%。结论可见免疫组化染色阳性率最高,具有比较好的检测显示效果,因此随着免疫组织化学技术的发展,未来会给检测幽门螺杆菌带来更大的发展和进步。     

Eosin staining of gelatine

Summary The mechanism of gelatine staining with four selected fluorone derivative dyes (eosin y, ethyl eosin, methyl eosin, uranin) was investigated. Gelatine films were stained in dye-buffer-ethanol solutions at varying pH and in the presence of NaCl and urea. Dye binding was recorded spectrophotometrically. Ionization constants of auxochromic phenolic groups were determined from pH-absorbance curves of dye-buffer-ethanol solutions. Dyebinding was greatest at pH below pK_OH and decreased with increasing pH. The addition of NaCl reduced dye binding slightly below pK_OH but markedly above pK_OH. The addition of 8 M urea decreased dyebinding regardless of pH. Comparing the pH dependence of dyebinding for eosin y and esterified eosins with ionization constants revealed that ionic bonding is unlikely to occur at the carboxyl group as well as at the phenolic group. Dye binding is intimately related to the presence of Br-groups. These results are discussed in conjunction with the functional structure of the dye ions and current concepts of dyebinding mechanisms.     

Microwave-stimulated immunogold silver staining

Summary The effect of microwave-generated energy for the demonstration of immunoglobulins in periodate lysine paraformaldehyde dichromate fixed paraffin sections of human reactive tonsil using the immunogold silver staining technique has been evaluated. Results of the study show that microwave stimulation permits incubation times of both the primary antibody and the immunogold reagent to be greatly reduced. Specific staining of equal intensity to that produced using the longer standard procedure together with minimal background staining is achieved.