Immunocytochemical identification of amyloid in formalin-fixed paraffin sections

Summary Formalin-fixed paraffin sections of livers, spleens and kidneys from patients with primary, secondary and familial amyloidosis as well as from a casein-induced murine amyloid model were analysed by an immunocy-tochemical (unlabeled antibody enzyme) method utilizing antisera to amyloid-related proteins. All amyloid deposits of all amyloid types showed positive reactions with anti-AP of the respective species. Positive reaction of anti-human AA to human secondary amyloid deposits and of anti-mouse AA to the deposits of casein-induced murine amyloid was also observed, but there was no species cross reactivity. No significant deposition of the reaction products was produced by anti-immunoglobulin light chains on deposits of any amyloid type, or by anti-AA in the tissues from primary or familial amyloidosis. The results indicate that amyloid proteins AA and AP can survive as antigens through routine histologic preparation, that anti-AP can be a universal marker for deposits of any amyloid type within the same species, and that AA-type amyloid can be identified by this method while there may as yet be no feasible universal marker for the AL-type at present.Presented in part at the 64th Annual Meeting of the Federation of American Societies for Experimental Biology, Anaheim, California, April, 1980     

Red-blue staining of hydrolysed nucleic acids in paraffin sections

A previous treatment with 10% HC1 in tetrahydrofuran for 2-3 min at 37° C hydrolyses DNA while substantially preserving RNA in formol-fixed paraffin sections. If this treatment is followed by dyeing with basic fuchsin-thiazine or oxazine mixtures, the basic fuchsin stains DNA, the blue dye cytoplasmic RNA, though nucleolar RNA is not well preserved. A specimen sequence is to treat the hydrolysed section with a mixture of 1% aqueous trimethylthionin (Chroma), 15 ml; 0.1% basic fuchsin (G. T. Gurr), 4 ml; and glacial acetic acid, 1 ml. Stain for 15-30 min, dehydrate in acetone, then pass sections through xylene to polystyrene. The specificity of this stain for cytoplasmic RNA is sharper than that of methyl green-pyronin; hence the technic given can be a useful addition to the standard Unna-Pappenheim procedure.     

Computer-aided image analysis applied to immunogold-silver staining: evaluation of proliferating cell nuclear antigen (PCNA)-reactive sites in paraffin sections

Feasibility of the combination of the immunogold-silver staining method (IGSS) and computer-aided image analysis was assessed for the detection of antigen in an immunostained, paraffin-embedded section. Using low-temperature IGSS, we stained a specimen of human oral squamous cell carcinoma with a monoclonal antibody, PC 10, against a proliferating cell nuclear antigen (PCNA/cyclin), and the section was analyzed by ACAS 570 interactive laser cytometry. The PCNA-positive cells, exhibiting a heteromorphic texture, were contrasted by the dark staining of their nuclei, but showed heterogeneity in staining intensity from cell to cell. Using a conventional microscope light source rather than a laser, and by employing the COMPLEMENT DATA program (which permits inversion of the data values) installed in the ACAS 570 software system, we were able to obtain a complemental image which replicated the real immunohisto-morphology. Approximately 30–35 cells from three different areas in the same section were selected by DEFINE CELL and MARK AREA programs, and quantitative image analysis was performed in terms of cell integrated value, area, perimeter, and shape factor indicated in histogram form. The combined utilization of IGSS with computer-aided image analysis was demonstrated to offer a crucial advantage for the quantitative assessment of immunostained sections.     

The potential of the immunogold-silver staining method for paraffin sections

Summary After perfusion fixation of the rat kidney with glutaraldehyde, and postfixation of the renal cortex with osmium-low ferrocyanide (40 mM OsO₄+6 mM K₄Fe(CN)₆ in 0.135 M phosphate buffer, pH 8.0), secondary lysosomes of proximal tubule cells carry acoat of electron dense material on the inner surface of the lysosomal membrane. This coat separates matrix and membrane of lysosomes, and corresponds in location and width to the electron translucent halo of conventionally processed lysosomes in TEM. The material which forms the coat, is stained by phosphotungstic acid at pH 0.3, and by periodic acid — thiocarbohydrazide — silver proteinate more intensively than the cell surface coat of the same cell; it contains a high concentration of hydroxyl,vicinal-glycol and α-aminoalcohol groups. Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

改良革兰染色法在显示石蜡切片真菌中的应用

目的建立真菌的病理切片改良革兰染色法。方法选取确诊真菌感染的组织蜡块,切若干空白片,通过苏木素-伊红染色（HE）、高碘酸-无色品红染色（PAS）、六胺银染色（GMS）、改良革兰染色后,比较改良革兰染色法的染色效果。结果改良革兰染色法的染色效果好,该法具有易操作、染色效果稳定等优点。结论改良革兰染色法能够清晰地显示出组织切片中的真菌,并且染色效果稳定,在检测组织中的真菌时可发挥重要的辅助诊断价值,具有广泛的应用前景。     

Combined histochemical staining of 1,2-glycol and sulfate groupings of mucopolysaccharides in paraffin sections

Summary A staining procedure has been developed for the simultaneous demonstration of 1.2-glycol and sulfate groupings of mucopolysaccharides in paraffin sections. It is a combined periodic acid-Schiff (PAS) and acriflavine technique, and by this technique the different groupings of mucopolysaccharides in several tissues of the rat and mouse have been stained. The results of a series of histochemical experiments on the dual staining reaction of the mucopolysaccharides have substantiated the reliability of the present technique.     

Improvement in the specificity of the silver staining technique for AgNOR-associated acidic proteins in paraffin sections

Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiff's reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.     

The influence of fixation on immunoperoxidase staining of plasmacells in paraffin sections of intestinal biopsy specimens

Summary The influence of different fixation methods on the results of immunoperoxydase staining of immunoglobulin and gastrin producing cells in gastric and duodenal mucosa was investigated. An indirect method was used on paraffin sections. It appeared that that fixatives containing sublimate gave the most consistent results, a sublimate-formaldehyde mixture being the best.     

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.