Hematoxylin Counterstain Research Articles

Gastric epithelial dysplasia.

Molecular abnormalities of the p.53 gene in chromosome 1 7p may be among the most commonly observed in human cancer. Their role in gastric carcinogenesis is suggested by their frequent detection in invasive adenocarcinomas. To investigate the chronology with which these abnormalities appear in the gastric carcinogenesis process, the expression of p53 proteins was investigated in late stages of the process, namely dysplasia, and in superficial carcinomas. A polyclonal antibody, CM-i, against both wild-type and mutant proteins was applied to paraffinembedded biopsy and gastrectomy specimens previously fixed in buffered formalin. Positive nuclear stain was obtained in 36.4% of 33 cases of gastric epithelial dysplasia, corresponding to 19% of mild, 27.3% of moderate, and 64.3% of severe dysplasias. Eight of 13 (61.5%) invasive carcinomas showed positive stain. The data indicate an increased incidence of p53 abnormalities in the late stages of gastric carcinogenesis. Introduction Deletions and insertions in the short arm of chromosome 1 7 as well as point mutations of the p53 gene have been reported frequently in human tumors, especially in the lung, breast, stomach, and colon (1-7). The nuclear proteins encoded by the p53 gene are suspected to play a regulatory role in the cell cycle. The normal protein has a short half-life, which makes it invisible in normal tissues by the usual immunohistochemical stains. Overexpression of the wild-type protein may occur abnormally in certain tumors (8). The proteins expressed by mutant p53 genes are more stable and more easily detected by immunochemical methods (9-11). Polycbonal antibody (CM-i) has been developed to detect both wild-type and Received 4/22/92. 1 Performed under the auspices of the R. Farini Foundation for Gastroenterological Research and NIH Grant P01-dA28842. 2 To whom requests for reprints should be addressed, at Department of Pathology, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70112. mutant p53 proteins utilizing pure soluble recombinant human p53. The CM-i antibody is able to detect protein in archived formalin-fixed and paraffin-embedded human tissues (12). Positivity of p53 protein by immunochemical methods has been reported in invasive gastric carcinomas and has been correlated with prognosis (13). Our interest has been in the precancerous gastric process, characterized by steps identifiable in tissue sections stained with hematoxylin and eosin. These steps involve atrophy, intestinal metaplasia, and dysplasia of the gastric mucosa (14). Archival material displaying several degrees of dysplasia as well as (predominantly superficial) invasive carcinomas was stained with CM-i polyclonal antibody to p53 proteins in order to investigate the timing of the abnormal expression of p53 proteins. Materials and Methods From a series of Italian patients enrolled in a prospective study of GED,3 24 subjects with a histological diagnosis of GED were selected. Each patient underwent multiple biopsies according to a protocol described in a previous study (15). In two of these patients, a gastrectomy was also performed because of the occurrence of GC. In addition, 9 cases of GED detected in stomachs resected for gastric cancer (8 cases) or for severe dysplasia (1 case) were also studied with extensive sampling of dysplastic and carcinomatous areas. All gastric biopsies as well as the surgical specimens were immediately fixed in buffered formalin (12 h for biopsy specimens, 24 h for resected stomachs). Fivetm-thick sections were stained with hematoxylin and eosin. Serial sections were used in the immunohistochemistry study. They were dewaxed in xylene (twice for 5 mm each) and rehydrated through serial alcohols (i00%, 95%; two changes of 3 mm each) to distilled water (twice for 5 mm each). Polyclonal antibody against both wild-type and mutant p53 protein (CM-i; Signet Laboratories, Inc., MA) (12) were used at a working concentration of 1 :25 at room temperature for 20 mm. Hydrogen peroxide, normal goat blocking serum, biotinylated immunoglobulins, avidin-biotin complex, and 3-amino-9-ethylcarbazole substrate solution were used according to the instruction from a commercial kit (Signet ELITE avidin-biotin detection system; Signet Laboratories, Inc.). Sections received a light Mayer’s hematoxylin counterstain, were mounted with aqueous mounting medium (Crystal/Mount; Biomeda, CA), and were postmounted with Postmounting Crystal/Mount in Permount (Biomeda). Positive control sections from for3 The abbreviations used are: GED, gastric epithelial dysplasia; Gd, gastric carcinoma. American Association for Cancer Research Copyright © 1992 on July 9, 2011 cebp.aacrjournals.org Downloaded from

Double and triple immunocytochemical labelling at the light microscope level in histopathology

Double and triple immunocytochemical detection methods for routine use in histopathology were investigated. For double immunostaining, the combinations of immunogold-silver staining (IGSS, black) with an immunoperoxidase method (3-amino-9-ethylcarbazole, red-brown) or with an immunoalkaline phosphatase method (Fast Red TR, red) proved very useful. These were followed by a Haematoxylin counterstain. An alternative approach using immunoperoxidase (red-brown) and immunoalkaline phosphatase (Fast Blue, BB, blue) methods was also successful, particularly for frozen sections of unfixed tissue and for the establishment of intermediate filament coexpression in tumours. The coexistence of desmin with vimentin in rhabdomyosarcoma, and of glial fibrillary acidic protein with vimentin in ependymoma, could be demonstrated directly by means of non-crossreacting murine and rabbit antibodies in the above combinations. The black (IGSS), red-brown (immunoperoxidase) and blue (immunoalkaline phosphatase) colours gave excellent contrast in triple immunostaining. The side-by-side demonstration of helper and suppressor T lymphocytes during renal allograft rejection, of kappa and lambda light chains in B-immunoblastic lymphoma, and of T and B lymphocyte populations in non-Hodgkin's lymphomas provided immediate information on the topography and cellular organization of the tissues.

A Solution for Removal of Resin from Epoxy Sections

Development of a resin-dissolving solution for use at low alkali concentrations is described. Crown ether dissolved in dimethyl-sulfoxide produces a superbasic alkoxide anion. A five minute treatment resulted in complete resin removal from kidney biopsy specimens embedded in Epon 812. Specimens were well stained by Loeffler's methylene blue. Periodic acid-methenamine silver and Giemsa stains yielded good results. Application of PAS reaction and subsequent hematoxylin counterstaining was practicable for diagnosis.

An Improved Immunoperoxidase Technique Using Horseradish Peroxidase Avidin D

We have compared the avidin-biotin complex immunoperoxidase method with a modified horseradish peroxidase a vidin D technique. Use of the horseradish peroxidase avidin D procedure saved time without compromising quality of the staining, by reducing the amount of time for methanol-hydrogen peroxide block, serum block, antibody incubations, horseradish peroxidase avidin D incubation, and Gill-3 hematoxylin counterstain, and cut costs by using higher dilutions of primary antibodies. Other helpful modifications include the utilization of phosphate-buffered saline, batching slides for secondary antibody and horseradish peroxidase avidin D, and instituting a standard development time.

Method for showing human spermatogenesis using Arachis hypogaea (AHA) lectin.

Using biotinylated Arachis hypogaea agglutinin (AHA) or peanut lectin (PNA) and an avidin-peroxidase procedure, the various stages of spermatid development were visualised with great clarity; a light haematoxylin counterstain permitted the easy recognition of spermatogenic cells in the same section. This method is particularly useful in the interpretation of poorly fixed material and may be helpful in studies of cyclical maturation of spermatozoa, irrespective of whether the material is obtained at biopsy or necropsy.

Histological observations on intrahepatocytic copper-containing granules in rainbow trout reared on diets containing elevated levels of copper

The histological nature of hepatic copper deposition in the liver of rainbow trout reared on diets containing elevated levels of copper was investigated. Two growth trials were conducted using practical diets with graded levels of supplemental copper, with determined maximum levels of 3088 mg/kg and 664 mg/kg copper. Histochemical observations using a rhodanine stain with a Harris hematoxylin counterstain revealed the presence of many rhodanine-positive granules in the hepatocytes of trout reared on diets containing the highest levels of copper supplementation. Ultrastructurally, electron-dense granules were visible in the cytoplasm of individual hepatocytes. Electron microprobe analysis confirmed the presence of large amounts of copper in these granules, along with a substantial amount of sulphur. These observations show that rainbow trout have the ability to sequester dietary copper in discrete granules in the cytoplasm of hepatocytes. The pericanalicular arrangement of the granules within the hepatocyte may suggest that these granules function in the excretion of excess copper in the bile.

Diagnosis of congenital neurogenic abnormalities of the bowel with monoclonal anti-neurofilament antibodies

The diagnostic properties of two monoclonal, antineurofilament antibodies were tested in a retrospective investigation of bowel specimens of 24 cases of Hirschsprung's disease, eight cases of long-segment aganglionosis, eight cases of pseudo-obstruction, and six cases of chronic constipation. Immunohistochemical staining with the antibodies in combination with hematoxylin counterstaining revealed six distinctive and divergent pictures, demonstrating innervation disturbances in all cases. Apart from Hirschsprung's disease and long-segment aganglionosis, characteristic pictures also appeared for pseudo-obstruction and, diversely, for chronic constipation. In addition, anomalies were revealed in the ganglionic, proximal bowel of five patients with Hirschsprung's and two with long-segment aganglionosis, who had all suffered from chronic constipation postoperatively.

Herpetic tracheobronchitis detected at bronchoscopy: cytologic diagnosis by the immunoperoxidase method.

Herpes simplex virus (HSV) infection of the respiratory tract requires rapid specific diagnosis to avoid late complications and maximize the efficacy of available drug therapy. We report a method of accomplishing this that was tested in 33 cytologic specimens derived from sputum, washings, or brushings from 25 debilitated elderly males suffering from a variety of underlying neoplastic and nonneoplastic chronic diseases. All specimens had shown either single cells (54%), multinucleated groups (8%), or both (38%); they displayed the ground-glass appearance (86%), discrete nuclear inclusions (4%), or both (10%), as appreciated by the Papanicolaou stain. The specimens were processed by the peroxidase-antiperoxidase technique utilizing anti-HSV-1 antibody and 3,3'-diaminobenzidine (DAB) as the chromogen. In six cases, aminoethylcarbazol (AEC) was used for comparison. Twenty-nine of the 33 specimens (88%) stained positively for HSV-1 as did control sections of herpetic encephalitis and esophagitis; there were no false positives with appropriate negative controls. All 12 bronchoscopic specimens revealed virocytes with HSV immunopositivity; in contrast, 17 of 21 (80%) sputum specimens were positive for HSV. The strongest positivity was noted in bronchial brushings and washings whereas the only four negative smears were from sputum specimens. The DAB immunostain was coarser and stronger at the periphery of the cytoplasm, and the hematoxylin counterstain permitted a clear identification of nuclear viral changes. With AEC, the immunostain was more vivid and evenly distributed, but counterstaining was impaired due to the greater solubility of the chromogen. The technique is sensitive, reproducible, and less expensive and time-consuming than electron microscopy (EM) or viral cultures.

Haematoxylin counterstaining of immunofluorescence preparations.

Haematoxylin counterstaining of immunofluorescence preparations.

Soil ameba infection. Specific indirect immunoenzymatic (peroxidase) staining of formalin-fixed paraffin sections.

This report describes the use of the immunoenzymatic (peroxidase) method to identify the species and to stain distinctively the amebas in formalin-fixed paraffin-mounted sections. This permits the use of hematoxylin and eosin counterstaining. The method, now well developed by others for many purposes, is an alternative to immunofluorescence and seems to offer a number of advantages and a lesser number of disadvantages.

A Histologic Study of the Developing Conduction System in the Turkey Heart

Hearts of embryonic turkeys, 10 days and older, and young poults, 1 day, 1 week, and 6 weeks old, were fixed in either cold (4°C.) 10% formalin or Rossman’s mixture. Adjacent sections, 7μ. in thickness, were stained with: (a) the periodic acid-Schiff technic of McManus with hematoxylin counterstain and salivary amylase controls, (b) hematoxylin and eosin, (c) Mallory’s triple stain, and (d) Movat’s pentachrome stain.The first histologic indication of conduction cells was seen in the hearts of 15-day embryos. These cells were seen both in the subendocardial region and perivascularly, especially in relation to the larger vessels present in the myocardium. Results indicate that conduction cells: (1) arise de novo, (2) occur extensively throughout the turkey heart, (3) are void of stainable glycogen, (4) are surrounded by a reticular connective tissue sheath, (5) possess striations, (6) are often binucleate, (7) are appreciably larger than the usual myocardial cells, and (8) usually are joined together to form fibers with no apparent intercalated discs separating the individual cells as in true myocardial fibers.