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At least 1,135 records · Page 63Linked to original sources
PubMed2005

Detection and Isolation of Mycobacterium avium subspecies paratuberculosis from intestinal mucosal biopsies of patients with and without Crohn's disease in Sardinia.

OBJECTIVES: Sardinia is an island community of 1.6 million people. There are also about 3.5 million sheep and one hundred thousand cattle in which Johne's disease and Mycobacterium avium subspecies paratuberculosis infection are endemic. The present study was designed to determine what proportion of people in Sardinia attending for ileocolonoscopy with or without Crohn's disease were infected with this pathogen. METHODS: Mycobacterium avium subspecies paratuberculosis was detected by IS900 PCR on DNA extracts of fresh intestinal mucosal biopsies as well as by isolation in culture using supplemented MGIT media followed by PCR with amplicon sequencing. RESULTS: Twenty five patients (83.3%) with Crohn's disease and 3 control patients (10.3%) were IS900 PCR positive (p = 0.000001; Odds ratio 43.3). Mycobacterium avium subspecies paratuberculosis grew in cultures from 19 Crohn's patients (63.3%) and from 3 control patients (10.3%) (p = 0.00001; Odds ratio 14.9). All patients positive by culture had previously been positive by PCR. Mycobacterium avium subspecies paratuberculosis first appeared in the liquid cultures in a Ziehl Neelsen (ZN) staining negative form and partially reverted through a rhodamine-auramine positive staining form to the classical ZN positive form. This resulted in a stable mixed culture of all 3 forms illustrating the phenotypic versatility of these complex chronic enteric pathogens. CONCLUSIONS: Mycobacterium avium subspecies paratuberculosis was detected in the majority of Sardinian Crohn's disease patients. The finding of the organism colonizing a proportion of people without Crohn's disease is consistent with what occurs in other conditions caused by a primary bacterial pathogen in susceptible hosts.

Adolescent↗
PubMed1970

Electron microscopic study of lipopolysaccharide from an avian strain of Escherichia coli O18.

The fine structure of lipopolysaccharide (LPS), isolated from an avian strain of Escherichia coli O18, was examined by electron microscopy. In positively stained preparations, ribbonlike structures with frequent branching were observed as previously reported (4). Two densely stained parallel lines were occasionally seen associated with a ribbon. When negative staining was employed, the LPS appeared as a branching ribbon with one central and two lateral zones divided by two relatively dense parallel lines running the complete length of the ribbon. The lateral zones were probably due to the O-antigenic side chains of the LPS. This interpretation was supported by the fact that the electron microscopic structure of the LPS from two rough strains, E. coli K-12 Gal 23 and Salmonella tuphimurium TV119 RII, both lacking the O-specific side chains, did not possess the outer lateral zones.

Centrifugation↗
PubMed2006

Neonatal Dubin-Johnson syndrome: long-term follow-up and MRP2 mutations study.

Neonatal Dubin-Johnson syndrome (DJS) is rarely diagnosed and mutational analysis of multidrug-resistance-associated protein 2 (MRP2) in such patients had not been reported. We aimed to investigate the possible correlations between genotype and phenotype of patients with DJS. Four cases of DJS, two diagnosed during the neonatal period and two diagnosed at adolescence, were followed for 5-20 y. Mutational analysis in the MRP2/ABCC2 gene was performed in all four cases. Biphasic pattern of jaundice attack was observed in one patient who was followed for 20 y, with jaundice subsiding before 1 y of age and recurring at adolescence. Six novel mutations in four patients were found, including deletions (2748del136, 3615del229, and Del3399-3400), and missense mutations (L441M and E1352Q) and nonsense mutation (Y1275X). The immunohistochemical staining in liver tissues from two patients with neonatal onset showed negative staining for MRP2. Reviewing previously reported cases, all patients diagnosed as DJS before 10 y of age have mutations involving one of the two ATP-binding cassettes (ABC) of the MRP2. This study suggests that long-term follow-up is indicated for neonatal DJS because of possible recurrence and/or second attacks of jaundice in later life, and that disruption of functionally important ABC domains in MRP2 may be related to the earlier onset of the disease.

Adolescent↗
PubMed1999

Glial heterotopia in the subcutaneous tissue overlying T-12.

Heterotopic glial nodules occur most commonly in the head and neck area, and are theorized to arise following abnormalities in the development of the facial and skull bone plates. However, in spite of the fact that some of these lesions are associated with communication with the central nervous system (CNS), the lack of a meningeal component, argues against simple herniation and separation of brain tissue through a defect in the skull. We present an infant with a nodule directly over the spine present in the T-12 region with no skin abnormalities. Magnetic resonance imaging (MRI) and computerized axial tomography (CT) showed no spinal abnormalities with an overlying fibrotic soft tissue mass. The patient had no other associated clinical findings. Histologic findings showed a cellular component arising within the reticular dermis with a deep circumscribed margin. The nodule contained irregularly shaped cells containing abundant cytoplasm and indistinct cellular margins with bland nuclei. These cells were clustered around and between a fibro-mucinous stroma. Immunohistochemical stains showed positive staining for S-100 protein, vimentin, GFAP, NSE, and CD57, and negative staining for Ki-67, CD34, Neurofilament protein, cytokeratin, and EMA. The spindle cells showed positive staining for CD34 and vimentin. The clinical and histologic features and immunohistochemical profiles are used to separate this lesion from the closely related, ependymal rests, ependymomas, and primary cutaneous chorodomas.

CD57 Antigens↗
PubMed2002

[Detection of human sperm morphology and acrosome reaction with Coomassie brilliant blue staining].

OBJECTIVES: To evaluate the application of Coomassie brilliant blue (CBB) G250 staining for the detection of human sperm deformity rate, rate of intact acrosome and acrosome reaction. METHODS: The smear of spermatozoa before and after capacitation and induced acrosome reaction with progesterone (P) were stained with 0.05% CBB G250 and Wright-Giemisa solution respectively, and visualized with light microscopy. The deformity rate of spermatozoa, rate of intact acrosome and acrosome reaction were calculated. RESULTS: There was no any difference in detection of deformity rate of spermatozoa and rate of intact acrosome with CBB G250 and Wright-Giemisa staining(P < 0.05). The sperm population of acrosome reaction with induced P was divided by CBB staining into two types: positive staining with dark violet blue on acrosome cap and pale or negative staining on the same area. The rate of the latter was increasing with increasing inductive time, maybe representative of the rate of acrosome reaction. The mean rate was(75.1 +/- 3.8)% after induced for 1 h. CONCLUSIONS: CBB G250 staining is a reliable method for assessment of the human sperm morphology and acrosome reaction.

Acrosome↗
PubMed2004

The morphological characteristics and expression of cell cycle regulatory proteins in cellular variants of idiopathic focal segmental glomerulosclerosis.

OBJECTIVE: To investigate the morphological characteristics and expression of cell cycle regulatory proteins in cellular variants of idiopathic focal segmental glomerulosclerosis (FSGS). METHODS: Seventeen cases of cellular variants of FSGS were studied by light microscopy, immunofluorescence (IF), and electron microscopy (EM). The immunohistochemistry and immunoelectron microscopy for the detection of cyclins (cyclin D1, cyclin E, cyclin A, cyclin B1) and cyclin dependent kinase inhibitors (CKIs, including p21, p27, p57) were performed in these cases. RESULTS: The hypertrophy and hyperplasia of epithelial cells overlying sclerotic or collapsed glomerular tufts were the prominent characteristics of cellular variants of FSGS; IF showed segmental deposits of IgM; hyperplastic epithelial cells possessed the features of both podocyte and parietal epithelial cells ultrastructurally. Hyperplastic epithelial cells of cellular lesions showed positive staining for cyclin E, cyclin A, cyclin B1 and p21, and negative staining for cyclin D1, p27 and p57. CONCLUSION: The hyperplastic epithelial cells in cellular variants of FSGS may be derived from damaged podocytes, which mimic the immature podocytes, re-engage the cell cycle to proliferate and form the cellular lesions. The up-regulation of cyclins (cyclin E, cyclin A, cyclin B1) concurrent with the loss of CKIs (p27, p57) contributes to the cell cycle regulation of cellular lesions of FSGS.

Adolescent↗
PubMedDate not supplied

Synchronous collecting duct carcinoma and papillary renal cell carcinoma: a case report and review of the literature.

The coexistence of multiple and synchronous primary neoplasms in the same organ (including kidney) has only rarely been described in the literature. We herein present a case of collecting duct carcinoma (CDC) combined with papillary renal carcinoma (RCC) having a 57-month disease-free survival CDC is a rather rare and aggressive neoplasm of the kidney. Sharing probably the same embryological origin, synchronous or metachronous association with in situ orpapillary transitional cell carcinoma (TCC) may be found; association with RCC has been only once reported in the literature. The high incidence of c-erbB-2 oncogene amplification in CDC further characterizes this tumor as a separate entity from renal cell carcinoma, and shows some genetic characteristics in common with TCC. The histological diagnosis of Bellini CDC can be confirmed by the positive immunohistochemical staining with a collecting duct marker and distal tubule marker and negative staining with a proximal tubule marker.

Aged↗
PubMed2004

Expression of matrix metalloproteinase-2 in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is a family of carcinomas arising from the renal tubular epithelium through different genetic lesions. The most common characterstics of RCC are hypervascularity, tendency to metastasize widely before giving rise to any symptoms or signs, and poor prognosis. It has been reported that matrix metallopropteinases (MMPs) are a family of endopeptidases implicated in tissue remodeling and cancer invasion. Therefore, the aim of the present study was to investigate the expression of MMP-2 in RCC and correlate its expression with the clinicopathologic prognostic parameters. MATERIAL AND METHODS: Fifty cases of primary RCC comprised the material of this study. Formalin-fixed paraffin-embedded tissues were stained with MMP-2 immunostaining, using improved streptavidin-biotin amplified system. The intensity of staining and percentage of positive cells were assessed. RESULTS: MMP-2 was expressed as diffuse brown cytoplasmic staining in positive cells. The non-tumorous renal tissue showed negative staining whereas the tumor cells, fibroblasts and vascular endothelial cells showed immunoreactivity in positive cases. 43/50 RCC cases studied were positive for MMP-2. A positive correlation was observed between MMP-2 expression and tumor size, histologic type, capsular & vascular invasion and high levels of cellular proliferation. CONCLUSION: It seems that MMP-2 is involved in tumor expansion phenomena associated with tumor progression, invasion of the microvasculature and distant metastasis of RCC.

Journal Article↗
PubMed1985

CAR-3, a monoclonal antibody-defined antigen expressed on human carcinomas.

Several monoclonal antibodies were raised against the human epidermoid carcinoma line A 431. The antibody produced by clone AR-3, when tested in enzyme-linked immunosorbent assay, was found to react with the cell line used as immunogen, the human gastric carcinoma line KATO III, the colon carcinoma line HT29, and the ovarian carcinoma line SW626. This monoclonal antibody was found unreactive when tested on human peripheral blood leukocytes or on a number of normal or neoplastic cell lines. The antibody precipitated a high-molecular-weight glycosylated component. When tested on paraffin sections by the avidin:biotin: peroxidase method, the AR-3 antibody stained pancreatic (6:7), gastric (11:14), ovarian (5:6), colon (4:8), endometrial (4:6), and cervical (4:7) carcinomas. A small minority of carcinomas of other organs was also stained. Sarcomas, lymphomas, and other tumors of nonepithelial origin were constantly negative. Staining of some normal epithelial cells was also observed. Among the fetal tissue tested, the antibody reacted with pancreatic ducts and the small intestine. The antibody recognized metastatic carcinoma cells in peritoneal effusions. On the basis of its tissue distribution, the antigenic determinant defined by the AR-3 monoclonal antibody was called CAR-3. The monoclonal AR-3 did not cross-react with partially purified preparations of carcinoembryonic antigen, gastrointestinal carcinoma antigen, or the human milk fat globule antigen. The AR-3 MAb appear, thus, to broaden the number of available reagents for histopathological diagnosis of carcinomas.

Animals↗
PubMed1987

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells.

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.

Animals↗
PubMedDate not supplied

Fine needle aspiration cytodiagnosis of leiomyosarcoma of the renal pelvis. A case report with immunohistochemical study.

Leiomyosarcoma of the renal pelvis is extremely rare. We report one such case correctly diagnosed by ultrasound-guided fine needle aspiration cytology (FNAC). The FNAC smears showed a monotonous population of closely packed, spindled tumor cells arranged in interlacing bundles. Those cells possessed markedly pleomorphic, hyperchromatic, elongated nuclei and a moderate amount of eosinophilic, fibrillary cytoplasm. Fascicles of similar spindle tumor cells arranged in a whorled pattern were seen in the section of the cell block. Immunohistochemistry was performed on the sections of the cell block preparation, showing intensely positive cytoplasmic staining of the tumor cells for muscle-specific actin but negative staining for CAM 5.2, AE1/AE3, S-100 protein and HMB-45. This staining pattern of the tumor allows its differentiation from other primary sarcomas of the kidney, sarcomatoid renal cell carcinoma, peripheral nerve sheath tumor and renal angiomyolipoma. The extreme degree of nuclear pleomorphism and hyperchromatism is not to be expected in renal leiomyoma or the usual cases of mesoblastic nephroma. Besides delineating the FNAC features of leiomyosarcoma of the renal pelvis, the present case illustrates the value of using the cell block preparation in FNAC.

Actins↗
PubMed1996

Malignant mesothelioma: immunohistochemistry and DNA ploidy analysis as methods to differentiate mesothelioma from benign reactive mesothelial cell proliferation and adenocarcinoma in pleural and peritoneal effusions.

OBJECTIVE: To determine whether malignant mesotheliomas can be differentiated from adenocarcinomas and benign reactive mesothelial cells in pleural and peritoneal fluids using immunohistochemical analysis in conjunction with DNA ploidy analysis. DESIGN: Sixteen cases of malignant mesothelioma, including epithelial, sarcomatous, and biphasic types, were collected. DNA analysis using flow cytometry and/or image analysis was performed on paraffin-embedded tissue from 15 of the mesothelioma cases, as well as on cytospin cell preparations from samples of pleural and peritoneal fluids from cases with either cytologically proven adenocarcinoma (seven cases) or benign reactive mesothelial cells (seven cases). Immunohistochemical studies were done in 15 mesotheliomas, 5 adenocarcinomas, and 4 benign reactive mesothelial cell effusions. RESULTS: All malignant mesotheliomas tested (100%) stained positively for prekeratin, whereas stains for carcinoembryonic antigen, B72.3, Leu-M1, and Ber-EP4 were negative. Stains vimentin, epithelial membrane antigen, and CA125 were positive in 75%, 75%, and 25% of cases tested, respectively. Benign reactive mesothelial cell cases stained similarly. Adenocarcinomas were more likely to react positively with B72.3, Ber-EP4, and carcinoembryonic antigen, and negatively with vimentin. DNA analysis showed that all benign cases were diploid, while all adenocarcinomas were nondiploid. Fifty-three percent of the malignant mesotheliomas were nondiploid. Sensitivity for detection of nondiploidy was greater for image analysis than for flow cytometry (100% vs 75%). CONCLUSIONS: B72.3, Ber-EP4, carcinoembryonic antigen, and vimentin are useful immunohistochemical markers in differentiating malignant mesotheliomas from adenocarcinomas, whereas immunohistochemistry does not reliably distinguish malignant from benign hyperplastic mesothelial cells. The addition of DNA ploidy studies is useful for differentiating the latter two groups.

Adenocarcinoma↗
PubMed1993

Immunohistochemical and nucleic acid analysis of somatotropin receptor populations in the bovine ovary.

Ovaries were analyzed for somatotropin receptor protein and mRNA through use of immunohistochemistry, solution hybridization/nuclease protection, Northern blotting, and reverse transcriptase polymerase chain reaction (RT-PCR). As indicated by immunoperoxidase staining, CL expressed immunoreactive somatotropin receptor (positive stain). Ovarian stroma, connective tissue, endothelium, and erythrocytes did not express somatotropin receptor (negative stain). Within the CL, somatotropin receptor protein was expressed primarily in large luteal cells whereas small luteal cells were negative. Most follicles (1-5 mm, after fixation) were negative for somatotropin receptor. On the basis of solution hybridization/nuclease protection, the mRNA for somatotropin receptor was found in greatest abundance in CL and large luteal cells and was nearly undetectable in small luteal cells or follicles (class 1, 3-5 mm; class 2, 6-9 mm; and class 3, > or = 10 mm). Northern blotting of mRNA for somatotropin receptor showed expression of somatotropin receptor mRNA transcripts in whole ovary (4.7 and 4.4 kb), CL (4.7 and 4.4 kb), and liver (4.4 kb); and RT-PCR amplified a single amino acid coding region for somatotropin receptor in CL and liver. In summary, somatotropin receptor (both immunoreactive protein and mRNA) is found primarily in the large luteal cell, and lesser amounts of the expressed receptor or its message are found in the follicle. Alternative sizes of mRNA for somatotropin receptor suggest novel mRNA processing in the bovine ovary.

Animals↗
PubMed2004

CDX-2, cytokeratin 7 and cytokeratin 20 immunohistochemical expression in the differential diagnosis of primary adenocarcinomas of the sinonasal tract.

Because the histopathological features of some primary adenocarcinomas of the sinonasal tract may show considerable overlap, we assessed the diagnostic value of a panel of immunohistochemical markers in the distinction between these malignancies. Paraffin-embedded tumour tissue sections from a series of 39 primary adenocarcinomas of the sinonasal tract, including 25 cases of intestinal-type adenocarcinoma (ITAC), 10 cases of salivary gland-type carcinoma and 4 cases of tubulopapillary low-grade adenocarcinoma were immunostained for CDX-2, cytokeratin 7 and cytokeratin 20. Diffuse nuclear staining for CDX-2 was identified in 80% of ITACs, while all non-ITACs were negative. Staining for cytokeratin 20 was positive in 84% of ITACs, including all cases negative for CDX-2, but negative in all other adenocarcinomas. Cytokeratin 7 was consistently positive in 88% of ITACs and in 100% of non-ITACs. Normal sinonasal epithelia expressed cytokeratin 7, but not CDX-2 and cytokeratin 20. Staining for CDX-2 and cytokeratin 20 has potential use in separating ITACs from other primary malignant glandular neoplasms of the nasal cavities and paranasal sinuses.

Adenocarcinoma↗
PubMed2002

Immunohistochemically detected hepatic micrometastases predict a high risk of intrahepatic recurrence after resection of colorectal carcinoma liver metastases.

BACKGROUND: Hepatic metastases from colorectal carcinoma frequently recur after resection and hepatic micrometastases most likely are important in the development of such recurrences. The objectives of the current study were to assess the feasibility of the immunohistochemical detection of hepatic micrometastases from colorectal carcinoma and to determine their clinical significance. METHODS: Fifty-three patients underwent curative hepatic resection for colorectal carcinoma metastases. Multiple tissue sections were cut from the advancing margin of the largest hepatic metastasis in each patient and were stained with an antibody against cytokeratin-20 to detect hepatic micrometastases, which were defined as discrete microscopic cancerous lesions surrounding the dominant metastasis. RESULTS: Normal hepatocytes and intrahepatic bile duct epithelia stained negative for cytokeratin-20 in all patients, whereas the largest hepatic tumors stained positive in 46 patients (86.8%). Among the 46 patients with hepatic tumors that were positive for cytokeratin-20, hepatic micrometastases were found immunohistochemically in 32 patients (69.6%). The presence of hepatic micrometastases was associated with a larger number of macroscopic hepatic metastases (P = 0.047) and patients with hepatic micrometastases were found to demonstrate a higher probability of intrahepatic recurrence (P = 0.003) compared with those patients without hepatic micrometastases. In addition, patients with hepatic micrometastases demonstrated a worse survival (10-year survival rate of 21.9%) compared with those patients without hepatic micrometastases (10-year survival rate of 64.3%) (P = 0.017). CONCLUSIONS: Immunohistochemical detection of hepatic micrometastases is feasible in patients with colorectal carcinoma liver metastases. Hepatic micrometastasis indicates widespread hepatic involvement and thus predicts an increased risk of intrahepatic recurrence after hepatic resection and a poorer patient prognosis.

Adult↗
PubMed2001

Short and mid-wavelength cone distribution in a nocturnal Strepsirrhine primate (Microcebus murinus).

Strepsirrhines are of considerable interest for understanding the evolution of cone photoreceptors because they represent the most ancestral living primates. The retina of nocturnal Strepsirrhines is reported to contain a single population of medium/long wavelength (MW/LW) cones whereas short wavelength (SW) cones are totally absent. The area centralis of nocturnal Strepsirrhines also lacks the degree of central specialization seen in the fovea of diurnal primates. In this study of a nocturnal Strepsirrhine, the gray mouse lemur (Microcebus murinus), we used specific antibodies that recognize SW and MW/LW opsins to determine the presence of different cone subtypes and their distribution in relation to that of rods and ganglion cells. The results are compared to two diurnal Haplorhine species, a New World (Callithrix jacchus) and an Old World (Macaca fascicularis) monkey. In the mouse lemur, both antibodies to MW/LW cone opsin (COS-1 and CERN956) label the same population of cones. A small proportion of SW cones is only stained by the JH455 antiserum whereas the monoclonal OS-2 antibody shows negative staining. These two antibodies label the same SW cone population in other primates. The extracellular matrix of all cones is also labeled by the peanut agglutinin (PNA) lectin. In mouse lemur retinal wholemounts, peak cone density is localized at the area centralis and ranged from 7,500 to 8,000 cones/mm(2). SW cones represent less than 0.2 % of the total cone population and are mainly located in the nasal part of the retina. SW cones show an irregular distribution and densities never exceed 49 cones/mm(2). The distribution of neurons in the ganglion cell layer shows a distinct centroperipheral gradient with a peak of 28,000 cells/mm(2) at the area centralis. Rod distribution shows a centroperipheral gradient with the peak (850,000 rods/mm(2)) including and extending slightly dorsal to the area centralis. The theoretical spatial resolution of the mouse lemur (4.9 cycles/degree) is slightly lower to that of other nocturnal primates. The densities of rods, cones, and ganglion cell layer neurons represent a compromise between spatial resolution and sensitivity for both photopic and scotopic vision.

Animals↗
PubMed1982

Malignant histiocytosis presenting as lethal midline granuloma: immunohistological study.

Recent advances in immunology have revealed that the "histiocytic" lymphoma of Rappaport is a proliferation of transformed lymphocytes rather than true histiocytes, and that the malignant cells in malignant histiocytosis (MH) are truly transformed histiocytes. We previously reported cases of MH presenting as lethal midline granuloma (MH-LMG) utilising fresh tissue specimens for the detection of cytological markers in a small number of cases. The results showed the true histiocytic nature of the proliferating cells. In this study, paraffin-embedded specimens from 16 cases with MH-LMG were processed for immunoperoxidase procedures to confirm the true histiocytic nature of the proliferating cells. Five cases with nasal and paranasal lymphomas (ML) were also stained to test the usefulness of immunoperoxidase procedures for distinguishing the MH-LMG from ML. Results were as follows; proliferating cells in MH-LMG were histiocytic in nature, staining positive for lysozyme in MH-LMG in contrast to the negative staining in ML.

Adolescent↗
PubMed1985

Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach.

A method involving rapid freezing followed by substitution fixation was developed, using acrolein as a fixative. This was then applied to several cytochemical stainings, and showed well preserved and clear cell structures. Membranes were apparently negatively stained and the ultrastructure of mitochondria, rough endoplasmic reticulum and Golgi apparatus was clearly discernible. The mitochondrial and cytoplasmic matrices were stained rather densely compared with routine chemically fixed preparations, implying a good preservation of matrix substances. Periodic acid-thiocarbohydrazide-silver proteinate staining was applied to the present method. The mucous granules of surface covering epithelial cells indicated fine staining of bipartite structure and the Golgi apparatus of mucous cells showed clear staining differences based on polarity. Postembedding lectin-ferritin and immunocytochemical stainings were applicable to the present preparations and stable stainings of secretory granules were obtained. A low temperature embedding material, Lowicryl K4M, was also examined. The cell preservation of these samples was not as good as those embedded in Epon, but the rough endoplasmic reticulum and Golgi apparatus of chief cells were stained with anti-pepsinogen antibody as were the secretory granules. The present method was also applicable to light microscopy.

Acrolein↗