scholarly journals Detection of Ehrlichia Risticii using an Avidin-Biotin-Immunoperoxidase Staining System

1989 ◽  
Vol 1 (3) ◽  
pp. 215-218 ◽  
Author(s):  
Mike A. Breider ◽  
Ginger Callahan ◽  
Richard E. Corstvet
Keyword(s):  
Enzymatic Digestion ◽  
Staining Procedure ◽  
Immunoperoxidase Staining ◽  
Formalin Fixed Paraffin ◽  
Ehrlichia Risticii ◽  
Formalin Fixed Paraffin Embedded ◽  
Indirect Immunoperoxidase ◽  
Neutral Formalin ◽  
Positive Results ◽  
Formalin Fixed

An indirect immunoperoxidase procedure using a specific anti- Ehrlichia risticii monoclonal antibody and an avidin-biotin-peroxidase staining method was used to detect E. risticii antigen in infected P388D1 murine monocytes. Several different methods of cytological fixation were used, including acetone (15 min), 95% ethanol (15 min), Bouin's fixative (5 hr), and 10% buffered neutral formalin (24 hr). The E. risticii organisms were labeled effectively and identified in cells fixed with acetone and ethanol. However, infected P388D1 cells fixed in 10% formalin or Bouin's fixative required enzymatic digestion with 1.0% trypsin for 15 min at 37 C before positive results were evident. This indirect immunoperoxidase avidin-biotin staining procedure proved to be a sensitive assay for the detection of intracellular E. risticii and may be an effective diagnostic procedure for formalin-fixed paraffin-embedded tissue.

A Standardized Staining Protocol for L1CAM on Formalin-Fixed, Paraffin-Embedded Tissues Using Automated Platforms

2014 ◽  
Vol 29 (2) ◽  
pp. 180-183 ◽  
Author(s):  
Mina Fogel ◽  
Ayelet Harari ◽  
Elisabeth Müller-Holzner ◽  
Alain G. Zeimet ◽  
Gerhard Moldenhauer ◽  
...  
Keyword(s):  
Cell Adhesion Molecule ◽  
Staining Procedure ◽  
Type I ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Staining Protocol ◽  
Automated Platform ◽  
Endometrial Carcinomas ◽  
L1 Cell Adhesion Molecule ◽  
Formalin Fixed

The L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers and can serve as a biomarker for prognosis in most of these cancers (including type I endometrial carcinomas). Here we provide an optimized immunohistochemical staining procedure for a widely used automated platform (VENTANA™), which has recourse to commercially available primary antibody and detection reagents. In parallel, we optimized the staining on a semi-automated BioGenix (i6000) immunostainer. These protocols yield good stainings and should represent the basis for a reliable and standardized immunohistochemical detection of L1CAM in a variety of malignancies in different laboratories.

scholarly journals Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue.

1988 ◽  
Vol 36 (5) ◽  
pp. 511-514 ◽  
Author(s):  
Y Hayashi ◽  
M Koike ◽  
M Matsutani ◽  
T Hoshino
Keyword(s):  
Human Brain ◽  
Enzymatic Digestion ◽  
Fixation Time ◽  
Reaction Products ◽  
Human Brain Tumor ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.

scholarly journals Detection of Mycobacterium Bovis in Formalin-Fixed, Paraffin-Embedded Tissues of Cattle and Elk by PCR Amplification of an IS6110 Sequence Specific for Mycobacterium Tuberculosis Complex Organisms

1997 ◽  
Vol 9 (3) ◽  
pp. 244-249 ◽  
Author(s):  
Janice Miller ◽  
Allen Jenny ◽  
Jack Rhyan ◽  
Dennis Saari ◽  
David Suarez
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Tuberculosis Complex ◽  
Rapid Diagnosis ◽  
Paraffin Sections ◽  
Tuberculosis Complex ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Positive Results ◽  
Formalin Fixed ◽  
Pcr Test

A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk ( Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex ( M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria ( M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.

scholarly journals Evaluation of three methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA by means of the PCR technique

2001 ◽  
Vol 15 (4) ◽  
pp. 314-318 ◽  
Author(s):  
Ricardo Alves MESQUITA ◽  
Evelyn K. ANZAI ◽  
Rogério Nogueira OLIVEIRA ◽  
Fábio Daumas NUNES
Keyword(s):  
Genomic Dna ◽  
Retrospective Studies ◽  
Enzymatic Digestion ◽  
Tissue Samples ◽  
Normal Oral Mucosa ◽  
Formalin Fixed Paraffin ◽  
Histopathologic Diagnosis ◽  
Formalin Fixed Paraffin Embedded ◽  
Digestion Methods ◽  
Formalin Fixed

There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

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