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Journal of Clinical Pathology Mar 2005The use in many countries of acid fixatives, such as Bouin's solution, has limited the use of archival tissue for molecular analysis. An acidic environment is one of the...
BACKGROUND
The use in many countries of acid fixatives, such as Bouin's solution, has limited the use of archival tissue for molecular analysis. An acidic environment is one of the main causes of DNA degradation. Moreover, RNA extraction is difficult in these types of fixed tissues.
AIMS
To amplify DNA and RNA from Bouin's fixed tissues.
METHODS
DNA and RNA were extracted from 20 breast cancer samples that had been routinely fixed in Bouin's fixative. Amplification of several genes using primers that produced amplicons of different lengths was carried out using the polymerase chain reaction (PCR) for DNA (with and without restoration) and reverse transcription PCR for RNA.
RESULTS
The acid environment of Bouin's fixative damaged both DNA and RNA. However, amplification was successful when the amplicon length was reduced to about 80 bp for RNA and 100-200 bp for DNA, especially if submitted to DNA reconstruction procedures.
CONCLUSIONS
It is possible to recover and analyse DNA and RNA from Bouin's fixed and paraffin wax embedded tissues.
Topics: Acetic Acid; Biological Specimen Banks; Breast Neoplasms; DNA Damage; DNA, Neoplasm; Female; Fixatives; Formaldehyde; Humans; Paraffin Embedding; Picrates; Polymerase Chain Reaction; RNA, Neoplasm; Reverse Transcriptase Polymerase Chain Reaction; Tissue Fixation
PubMed: 15735167
DOI: 10.1136/jcp.2004.016477 -
Ticks and Tick-borne Diseases Jul 2023Ticks are important ectoparasites that are capable of transmitting multiple classes of pathogens and are currently linked with many emerging tick-borne diseases...
Ticks are important ectoparasites that are capable of transmitting multiple classes of pathogens and are currently linked with many emerging tick-borne diseases worldwide. With increasing occurrences of tick-borne diseases in both humans and veterinary species, there is a continuous need to further our understanding of ticks and the pathogens they transmit. Whole tick histology provides a full scope of the tick internal anatomy, allowing researchers to examine multiple organs of interest in a single section. This is in contrast to other techniques that are more commonly utilized in tick-borne disease research, such as electron microscopy and light microscopy of individual organs. There is a lack of literature describing a practical technique to process whole tick histologic sections. Therefore, the current study aims to provide researchers with a workable protocol to prepare high quality paraffin-embedded whole tick histology sections. Amblyomma americanum adults were used as an example species for this study. After a series of pilot experiments using a combination of various fixatives, softening agents and processing techniques, we elected to compare two common fixatives, 10% neutral-buffered formalin (NBF) and Bouin's solution for whole ticks. Equal numbers of A. americanum unfed adults (n = 10/fixative) were processed identically and their whole tick histology coronal sections were individually scored. Higher scores were assigned to whole tick sections that contained more internal organs that are crucial for tick-borne disease research (e.g. salivary glands and midgut), high integrity of tissues and exoskeleton on the section, and good fixation and staining quality of the tissues. The mean total scores for Bouin's-fixed ticks were significantly higher compared to NBF-fixed ticks (p = 0.001). To further assess our preferred technique, we also demonstrated the feasibility of producing high quality whole tick sections for three other common tick species of medical importance (Rhipicephalus sanguineus, Ixodes scapularis, and Dermacentor variabilis) using Bouin's solution. While this technique may require further optimization for other tick species, we described a feasible protocol that uses commonly available tools, reagents and standard histologic equipment. This should allow any investigator to easily make adjustments to this protocol as needed based on their experimental goals.
Topics: Animals; Humans; Fixatives; Paraffin Embedding; Ixodes; Tick-Borne Diseases
PubMed: 36965259
DOI: 10.1016/j.ttbdis.2023.102162 -
Journal of Advanced Veterinary and... Jun 2022Rodlet cells produce secretions of glycoproteins in nature. This study investigated the microscopic morphology, histochemical and immunohistochemical reactions, and...
OBJECTIVE
Rodlet cells produce secretions of glycoproteins in nature. This study investigated the microscopic morphology, histochemical and immunohistochemical reactions, and distribution of the rodlet cells in the gut of Binni fish (Mesopotamichthys sharpeyi).
MATERIALS AND METHODS
Thirty samples were obtained from the cranial, middle, and caudal portions of Binni intestine immediately after being euthanized, fixed in Bouin's solution for 18 h at 24°C, and had undergone routine histological processing, different conventional histochemical stains, and immunostaining with TNF-α and S100 protein antibody.
RESULTS
The intestine of Binni fish showed different stages of rodlet cells classified into three distinctive forms: vesicular, granular, and mature cells. Rodlet cells are poorly stained with hematoxylin and eosin. Their secretory granules have a weak positive reaction with periodic acid-Schiff (PAS) and Alcian blue (AB), and react positively to combined AB and PAS. Rodlet cells were stained lightly with Safranin O, observed pink in color by Giemsa stain, and showed reactivity to Masson's and Mallory trichrome stains. Rodlet cells were immunostained positively against TNF-α and S100 antibodies, indicating that they have an immune function.
CONCLUSIONS
Rodlet cells, with their neutral glycoprotein secretions, play a crucial role in the immunity of Binni fish intestine.
PubMed: 35891664
DOI: 10.5455/javar.2022.i594 -
International Journal of Molecular... Sep 2019In clinical practice, patients' tissues are fixed and paraffin-embedded in order to enable histological diagnosis. Nowadays, those tissues are also used for molecular...
In clinical practice, patients' tissues are fixed and paraffin-embedded in order to enable histological diagnosis. Nowadays, those tissues are also used for molecular characterization. Formalin is the most used fixative worldwide, and Bouin's solution in some worldwide institutions. Among molecular targets, micro RNAs (miRNAs), the single-stranded non-coding RNAs comprised of 18 to 24 nucleotides, have been demonstrated to be resistant to fixation and paraffin-embedding processes, with consequent possible application in clinical practice. In the present study, , , , , , , and were investigated in formalin and matched Bouin's solution-fixed tissues of high grade serous ovarian cancers by means of real-time and droplet digital PCR (ddPCR). Micro RNAs were detectable and analyzable in both formalin- and Bouin's-fixed specimens, but on average, higher Ct values and lower copies/µL were found in Bouin's-fixed samples. Data from formalin-fixed samples correlated significantly for most targets with Bouin's ones, except for and . This study shows that miRNAs are analyzable in both formalin- and Bouin's-fixed specimens, with the possibility, after proper data normalization, to compare miRNA-based data from formalin-fixed samples to those of Bouin's-fixed ones.
Topics: Humans; Immunohistochemistry; MicroRNAs; Paraffin Embedding; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Tissue Fixation
PubMed: 31569791
DOI: 10.3390/ijms20194819 -
Journal of Clinical Pathology Nov 1994Methodological modifications, particularly the use of different fixatives, may account for discrepancies between studies of the relation between virulence and biofilm... (Comparative Study)
Comparative Study
Methodological modifications, particularly the use of different fixatives, may account for discrepancies between studies of the relation between virulence and biofilm production in vitro by isolates of coagulase negative staphylococci. The efficacy of formalin and Bouin's reagent for fixing coagulase negative staphylococcal biofilms in a microtitre tray assay was compared. The optical density of stained adherent growth by three strains was reduced by an average of 20% following fixation with 10% formaldehyde compared with Bouin's reagent. This difference seemed to be mainly because of increased background staining and blackening of the biofilm when Bouin's reagent was used. Formalin fixation was also effective at identifying early and late biofilm production in adherence growth kinetic experiments with 10 coagulase negative staphylococcal clinical isolates.
Topics: Acetates; Acetic Acid; Bacteriological Techniques; Biofilms; Coagulase; Fixatives; Formaldehyde; Picrates; Staphylococcus
PubMed: 7829683
DOI: 10.1136/jcp.47.11.1044 -
New Biotechnology Nov 2022Most tissues in clinical practice are formalin-fixed and paraffin-embedded for histological as well as molecular analyses. The reproducibility and uniformity of...
Most tissues in clinical practice are formalin-fixed and paraffin-embedded for histological as well as molecular analyses. The reproducibility and uniformity of molecular analyses is strictly dependent on the quality of the biomolecules, which is highly influenced by pre-analytical processes. In this study, the effect of different fixatives was compared, including formalin, Bouin's solution, RCL2® and TAG-1™ fixatives, by stringent application of ISO standards in mouse liver tissue processing, including formalin-free transport of tissues and tissue grossing in a refrigerated environment. The effect of fixatives was studied in terms of nucleic acid quality at the time of tissue processing and after one year of tissue storage at room temperature in the dark. Furthermore, a microcomputed tomography (CT) scan analysis was applied to investigate the paraffin embedding. The results show that the application of ISO standards in tissue processing allows analysis of 400 bases amplicons from RNA and 1000 bases from DNA, even in extracts from formalin-fixed and paraffin-embedded tissues. However, after one year storage at room temperature in the dark, a degradation of the nucleic acids was observed. Nevertheless, extracts can still be analyzed, but for metachronous tests it is highly recommended to repeat the quantitation of housekeeping genes in order to standardize the extent of nucleic acid degradation.
Topics: Animals; Fixatives; Formaldehyde; Mice; Nucleic Acids; Reference Standards; Reproducibility of Results; Tissue Fixation; X-Ray Microtomography
PubMed: 35878783
DOI: 10.1016/j.nbt.2022.07.001 -
Animal Reproduction 2021is endemic species in Indonesia. Study about fetal development of are very rare because of sample limitation. This study was carried out to describe the morphometrics...
is endemic species in Indonesia. Study about fetal development of are very rare because of sample limitation. This study was carried out to describe the morphometrics and x-ray analysis of three fetuses in different stage to give basic information about fetal development of . Three fetus samples fixed in Bouin's solution was used in this study. Observation was carried out to identify the characteristic of three fetus samples. This included the pattern of hair, body measurements, body volume, and body weight. X-ray analysis was carried out to know the ossification process in the fetal development. Statistical analysis was carried out using Microsoft 365® Excel program software. Three fetus samples had different specific hair pattern, that was hairless, smooth hairs, and smooth hairs with dense-non dense pattern. Body volume of 1, 2, and 3 fetus were 23mL, 90mL, and 170mL, respectively. Body weight of 1, 2, and 3 fetus were 19.5g, 79.22g, and 153.18g, respectively. Pearson's correlation analysis shown strong relationship between total body length, front body length, back body length, horizontal body diameter, vertical body diameter, head length, and head diameter against body volume and body weight of three fetuses. Significant positive correlation was shown between horizontal body diameter, vertical body diameter, and head diameter against body volume and body length with P value < 0.05. Faint radiopaque images showed in the 2 fetus sample and strong radiopaque images showed in the 3 fetus sample. Radiopaque images were identified in the teeth, cranium, vertebrae, and extremities bones. In this study we concluded that there was a specific hair pattern in different fetal stage. All body measurements have positive correlation with body volume and body weight and x-ray analysis shown that the ossification of the bone was started to happen while the smooth hair was growth.
PubMed: 34691263
DOI: 10.1590/1984-3143-AR2021-0005 -
The Journal of Histochemistry and... Jan 2020Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. The spatial-temporal localization of HA can be visualized in situ using biotinylated HA binding...
Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. The spatial-temporal localization of HA can be visualized in situ using biotinylated HA binding proteins (HABPs). This assay is sensitive to fixation conditions, and there are currently no best practices for HA detection. Thus, the goal of this study was to optimize fixation conditions for visualizing HA in the ovary, kidney, and liver through analysis of six commonly used fixatives for HA detection: Bouin's Solution, Carnoy's Solution, Ethanol-Formalin-Glacial Acetic Acid (EFG), Histochoice, Modified Davidson's Solution, and 10% Neutral Buffered Formalin. Organs were harvested from CB6F1 mice and fixed with one of the identified fixatives. Fixed organs were sectioned, and the HABP assay was performed on sections in parallel. Hematoxylin and eosin staining was also performed to visualize tissue architecture. HABP signal localization and intensity varied between fixatives. EFG and Carnoy's Solution best preserved the HA signal intensity in the ovary and liver, showing HA localization in various sub-organ structures. In the kidney, only Modified Davidson's Solution was less than optimal. Our findings demonstrate that fixation can alter the ability to detect HA in tissue macro- and microstructures, as well as localization in a tissue-specific manner, in situ.
Topics: Animals; Female; Humans; Hyaluronic Acid; Kidney; Liver; Mice; Organ Specificity; Ovary; Rats; Tissue Embedding; Tissue Fixation
PubMed: 31714169
DOI: 10.1369/0022155419884879 -
Infection and Immunity Dec 2017Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated...
Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated with -engineered strains isolated from equine uterine infections. Following establishment of infection, the horses were euthanized and the endometrial surfaces were imaged for luminescence to localize adherent -labeled bacteria. Samples from the endometrium were collected for cytology, histopathology, carbohydrate analysis, and expression of inflammatory cytokine genes. Tissue-adherent bacteria were present in focal areas between endometrial folds (6/6 mares). The Pel exopolysaccharide (biofilm matrix component) and cyclic di-GMP (biofilm-regulatory molecule) were detected in 6/6 mares and 5/6 mares, respectively, from endometrial samples with tissue-adherent bacteria ( < 0.05). A greater incidence ( < 0.05) of Pel exopolysaccharide was present in samples fixed with Bouin's solution (18/18) than in buffered formalin (0/18), indicating that Bouin's solution is more appropriate for detecting bacteria adherent to the endometrium. There were no differences ( > 0.05) in the number of inflammatory cells in the endometrium between areas with and without tissue-adherent bacteria. Neutrophils were decreased ( < 0.05) in areas surrounding tissue-adherent bacteria compared to those in areas free of adherent bacteria. Gene expression of interleukin-10, an immune-modulatory cytokine, was significantly ( < 0.05) increased in areas of tissue-adherent bacteria compared to that in endometrium absent of biofilm. These findings indicate that produces a biofilm in the uterus and that the host immune response is modulated focally around areas with biofilm, but inflammation within the tissue is similar in areas with and without biofilm matrix. Future studies will focus on therapeutic options for elimination of bacterial biofilm in the equine uterus.
Topics: Animals; Biofilms; Endometritis; Endometrium; Female; Genes, Reporter; Horse Diseases; Horses; Luciferases; Pseudomonas Infections; Pseudomonas aeruginosa
PubMed: 28970274
DOI: 10.1128/IAI.00332-17 -
Journal of Clinical Pathology Mar 2005To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin's fixed prostate biopsies.
AIMS
To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin's fixed prostate biopsies.
METHODS
Archival Bouin's fixed and formalin fixed, paraffin wax embedded prostatic biopsies were immunostained for three nuclear biomarkers (minichromosome maintenance protein 2 (MCM-2), p27, and Ki-67), one membrane localised biomarker (C-erb-B2), CD34, and alpha methylacyl-CoA racemase (AMACR). The quality of IHC staining was compared between tissues prepared separately in both fixatives. Feulgen staining was also performed on Bouin's fixed tissues to check its suitability for nuclear morphometry.
RESULTS
MCM-2 staining was completely negative in Bouin's fixed tissues, whereas p27 showed more background and excess cytoplasmic staining in Bouin's fixed versus formalin fixed tissues. C-erb-B2 showed non-specific, strong luminal cell staining in the Bouin's fixed tissue. Feulgen staining was also very weak in Bouin's fixed tissue. However, Ki-67, AMACR, and CD34 worked equally well in Bouin's and formalin fixed tissues.
CONCLUSIONS
Bouin's fixed tissues may be unsuitable when subsequent IHC and morphometry are contemplated. An awareness of which antibodies are suitable for use in Bouin's fixed biopsies is essential.
Topics: Acetic Acid; Biomarkers, Tumor; Biopsy; Cell Cycle Proteins; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p27; Fixatives; Formaldehyde; Humans; Male; Minichromosome Maintenance Complex Component 2; Neoplasm Proteins; Nuclear Proteins; Paraffin Embedding; Picrates; Prostatic Neoplasms; Receptor, ErbB-2; Rosaniline Dyes; Tissue Fixation; Tumor Suppressor Proteins
PubMed: 15735170
DOI: 10.1136/jcp.2004.019299