Neutral-buffered Formalin Research Articles

Characterization of high risk human papilloma virus genotypes associated with oropharyngeal cancers in a Nigerian population.

Introductionhead and neck cancers have essentially been a disease of the elderly but recent studies are beginning to demonstrate their increasing incidence in young people with infections such as human papilloma virus (HPV). This study was carried out to determine the prevalence of high risk Human papilloma virus (hrHPV) related oropharyngeal carcinoma and its prevalent genotypes as well as their strength of association with HIV in adult Nigerian subjects.Methodsthis was a cross-sectional study of 41 patients with oropharyngeal carcinomas seen over a 2-year period. Patients had incisional and/or excisional biopsy done under anesthesia. A portion of the specimen from which the DNA was extracted was placed in Digene HC2 DNA collection device while the 2nd portion for histopathological analysis was fixed using 10% Neutral Buffered Formalin (NBF) and embedded in paraffin blocks. Oropharyngeal cancer HPV genotyping was done using HPV genotypes 14 real-tm quant kit (SACACE, Italy). The data was analyzed using SPSS version 23.Resultsprevalence of HPV was 17.1% with a male to female ratio of 2.7: 1. The identified genotypes were 16, 33, 35 and 52 with 28.6% of patients having more than one genotype. Most of the age groups studied were affected. Squamous cell carcinoma and ameloblastic carcinoma were the cancers associated with HPV. HPV was not identified in the HIV positive patients.Conclusionhigh-risk human papilloma virus genotypes 16, 33, 35 and 52 are associated with oropharyngeal carcinoma in Nigeria but were not found in HIV patients. This finding provides a strong evidence for the use of the 9-valent prophylactic vaccine for the prevention of oropharyngeal cancer in Nigeria. Public awareness and HPV prevention strategies should reduce significantly the incidence of oropharyngeal carcinomas in our environment.

Graded doses of grape seed methanol extract attenuated ataxic movement, cerebellocortical toxicity, and impairment following chronic carbamazepine treatment in male Wistar rats

This study investigated the effects of chronic cotreatment of carbamazepine (CBZ) with grape seed methanolic extract (GSME) on the markers of neurotoxicity and motor coordination in male rats. Thirty male Wistar rats were randomized into 5 groups (n = 6) and treated orally with propylene glycol (PG 0.1 ml/day), CBZ (25 mg/kg), CBZ (25 mg/kg) + GSME (200 mg/kg), CBZ (25 mg/kg) + GSME (100 mg/kg), or CBZ (25 mg/kg) + GSME (50 mg/kg) for 28 days. Thereafter, the animals were subjected to motor-coordination tests and, eventually, sacrificed by cervical dislocation. The cranium was opened and the brain excised. The prefrontal cortex (PFC) and cerebellum were homogenized for the biochemical assessment, while representative brain was fixed in 10% neutral-buffered formalin for the histomorphological investigation. The results were presented as mean ± SEM, analyzed using one-way analysis of variance (ANOVA) and Student-Newman–Keuls post hoc analysis where appropriate, while p < 0.05 was considered statistically significant. Indices of motor coordination were significantly (p = 0.0014) impaired with a significant (p = 0.0001) increase in the concentration of malondialdehyde (MDA) in the PFC and cerebellar tissue. In addition, the activities of glutathione increased (p = 0.0001) significantly in the CBZ + GSME-treated rats. All these anomalies were attenuated in the CBZ + GSME treated rats.Coadministration of GSME with CBZ may ameliorate CBZ-induced neurotoxicity, histoarchitectural disorganization of PFC and cerebellum with resultant effect on fine motor actions.

Processing and Sectioning Undecalcified Murine Bone Specimens.

Preparation of mineralized tissue specimens for bone-specific staining encompasses a critical sequence of histological techniques that provides visualization of tissue and cellular morphology. Bone specimens are fixed in 10% neutral buffered formalin (NBF), dehydrated in graded ethanol (EtOH) solutions (and optionally cleared in xylene), infiltrated and embedded in polymethyl methacrylate (methyl methacrylate or MMA), classically sliced into 4-10 micrometer (μm) sections, and stained with bone-specific histological stains such as von Kossa (with either nuclear fast red solution counterstain or MacNeal's tetrachrome counterstain), modified Goldner's trichrome, Alizarin Red S, Safranin O, and tartrate-resistant acid phosphatase (TRAP) stain. Here, we describe the tissue processing of mineralized mouse bones from dissection to staining for histological analysis by light microscopy.

Glyoxal: a proposed substitute for formalin in H&E and special stains

ABSTRACT Neutral-buffered formalin (NBF) has been used as the primary fixative in anatomic pathology laboratories for decades. Although it yields excellent morphologic and staining results, NBF poses significant health hazards requiring tissue to be grossed under a grossing/chemical fume hood. Glyoxal fixatives offer far less toxic alternatives and do not necessitate use of a grossing hood. Using freshly extracted canine and feline testes, ovaries, and uteri, the effects of glyoxal and NBF fixation were compared. While NBF is still considered the gold standard, some glyoxal fixatives perform as well as NBF in regards to morphology, H&E staining properties, and histochemical staining properties.

Hypothalamic-pituitary-testicular axis derangement following chronic phenytoin-levetiracetam adjunctive treatment in male Wistar rats.

The effects of phenytoin (PHT), levetiracetam (LEV) or their adjunctive treatment on the hypothalamic-pituitary-testicular axis in male Wistar rats were determined. Twenty-eight male rats (150-180g) were randomised into four groups (N=7). Groups I to IV received intraperitoneal treatment of either normal saline (0.2ml i.p) or PHT (50mg/kg i.p) or LEV (50mg/kg) or PHT (25mg/kg) and LEV (25mg/kg) combination for 28days. The organ weight, concentrations of reproductive hormones and semen profile were determined, while the brain, epididymis and testis were preserved in 10% neutral-buffered formalin for the histomorphological study. Data were analysed using descriptive and inferential statistics. The results were presented as mean±SEM in graphs or tables, while the level of significance was taken at p<.05. The semen profile was altered significantly in all the treatment groups. The gonadotrophic releasing hormone, luteinising hormone and testosterone concentration decreased significantly in the PHT- and PHT+LEV-treated groups compared with control. There were various disruptions in the hypothalamus, pituitary and testis of the PHT- and PHT+LEV adjunctive-treated rats. In conclusion, chronic PHT+LEV treatment may pose deleterious effects on the hypothalamic-pituitary-testicular axis than single treatment of either of the two drugs in male rats.

Hydrogen peroxide as an oxidizing agent for glycogen demonstration using Schiff’s reagent in rabbit liver fixed with alcoholic and aqueous fixatives

ABSTRACT This study investigated the optimal concentration of hydrogen peroxide (H2O2) for demonstrating glycogen in rabbit liver using Schiff’s reaction as compared to the conventional periodic acid-Schiff (PAS) method. Four fixatives, Bouin’s, neutral buffered formalin (NBF), 80% ethanol, and Rossman’s, were used at room temperature (RT) and 4°C. Paraffin sections of rabbit liver (n = 192) were stained using 0.5%, 1% and 2% periodic acid-Schiff (PAS) and 0.5%, 1%, and 2% hydrogen peroxide (H2O2)-Schiff methods. It was found that 0.5% H2O2-Schiff provided good staining results, while 1% PAS produced excellent results for glycogen fixed with 80% ethanol at RT. Also, 0.5% H2O2-Schiff produced good staining of glycogen fixed with 10% NBF at RT as compared to 0.5% PAS which provided satisfactory staining results. Fixation with 80% ethanol at RT produced the best staining quality for glycogen using both oxidizing agents at the optimal concentrations of either 1% periodic acid or 0.5% H2O2.

Determination of anti-mullerian hormone in serum and ovarian histopathology of buffaloes

Present study was undertaken to assess AMH concentration in serum and histopathological study of AFC in buffalo ovaries. Total 30 pair of ovary were collected in 10% Neutral Buffer Formalin. Blood samples were also collected before slaughtering the buffalo animals for estimation of AMH concentration. Before processing, 10 serial sections of each pair of ovary was done for histopathological examinations. All the serial sections of the ovaries were processed as per the standard procedure. On the basis of histopathological examinations and follicle count, animals were categorized as cyclic and anestrus. Out of 30 pairs of ovary, 18 pairs were identified as anestrus and 12 as cyclic. The mean number of primordial, antral and growing ovarian follicles count of cyclic buffaloes were 0.42±0.07, 0.15±0.03 and 4.47±0.03 and 0.49±0.02, 0.16±0.03 and 0.41±0.06 for anestrus buffaloes, respectively. Growing follicles were significantly higher in cyclic as compared to anestrous buffaloes. However, the difference of primordial and antral follicle count remained non-significant in cyclic and anestrous buffaloes. Whereas, AMH concentration in cyclic buffaloes (273.50±48.52) was significantly higher than anestrous buffaloes (79.40±7.80). Therefore, it is concluded that, anti-mullerian hormone is an endocrine marker and can predict the fertility of animal. Also, follicular count is positively correlated with AMH concentration.

Effects of various fixatives and temperature on the quality of glycogen demonstration in the brain and liver tissues

The visualization of glycogen deposits in cells and tissues is important for studying glycogen metabolism as well as diagnosis of glycogen storage diseases. Evidence suggests that the demonstration of glycogen can better be enhanced by factors such the choice of fixative and temperature during fixation. Here, we assessed efficacy of neutral buffered formalin (NBF), alcoholic formalin (AF) and paraformaldehyde (PFA) at 4 °C, 37 °C and 40 °C using Periodic Acid Schiff's staining method. Each liver specimen was fixed in NBF and AF while the brain tissues were fixed in NBF, AF and PFA. We found that there was a better PAS staining intensity with the liver tissues fixed in AF compared with NBF. Also, there was no difference in the quality of the staining for tissues fixed in AF at 37 °C, 4 °C and 40 °C, but fixation with NBF at 4 °C gave the best staining quality when compared with 40 °C and 37 °C. Furthermore, hippocampal tissues fixed in AF showed better quality of PAS staining compared with NBF and PFA. A significant increase in staining intensity was observed for PFA when compared with NBF. Superior staining intensity for PAS was observed at 4 °C for hippocampal tissues fixed with NBF, AF and PFA. Taken together our results show that AF at a temperature of 4 °C gave the best result. Hence, glycogen demonstration can better be enhanced by the choice of fixative and temperature during fixation.

The diagnostic utility of cell block in fine needle aspiration cytology of palpable breast lesions in a Nigerian tertiary health institution.

Although fine needle aspiration cytology (FNAC) is highly accurate for detecting breast malignancies, concerns remain among cytopathologists about false-positive and false-negative diagnoses. Cell block (CB) preparations have been advocated by some cytopathologists as one of the methods to improve and consolidate the diagnostic accuracy of FNAC. The aim of this study was to determine the diagnostic utility of CB in FNAC of palpable breast lesions among female patients. Following FNA, CBs were prepared using 10% neutral-buffered formalin from the residual breast aspirates of 100 consecutive female patients attending the FNAC clinic. The slides of the conventional smears, CB and excisional biopsies were examined, and results were analysed using the SPSS. Of the 100 patients that had FNAC, 44 (44%) had excisional biopsy performed. An additional 13% diagnostic yield for malignancy was obtained with the use of CB preparations. CB reduced equivocal diagnoses by 25%, corresponding with 90.9% improvement on definitive diagnoses. In our setting, the addition of CB to smear remarkably improved the diagnostic utility of breast FNAC by minimising atypical and suspicious for malignancy diagnostic categories.

The effects of tissue fixation on sequencing and transcript abundance of nucleic acids from microdissected liver samples of smallmouth bass (Micropterus dolomieu).

There is an increasing emphasis on effects-based monitoring to document responses associated with exposure to complex mixtures of chemicals, climate change, pathogens, parasites and other environmental stressors in fish populations. For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology. Consequently, formalin-fixed, paraffin-embedded (FFPE) tissue can be an important reservoir of nucleic acids as technologies emerge that utilize molecular endpoints. Despite the cross-linking effects of formalin, its impact on nucleic acid quality and concentration, amplification, and sequencing are not well described. While fresh-frozen tissue is optimal for working with nucleic acids, FFPE samples have been shown to be conducive for molecular studies. Laser capture microdissection (LCM) is one technology which allows for collection of specific regions or cell populations from fresh or preserved specimens with pathological alterations, pathogens, or parasites. In this study, smallmouth bass (Micropterus dolomieu) liver was preserved in three different fixatives, including 10% neutral buffered formalin (NBF), Z-Fix® (ZF), and PAXgene® (PG) for four time periods (24 hr, 48 hr, seven days, and 14 days). Controls consisted of pieces of liver preserved in RNALater® or 95% ethanol. Smallmouth bass were chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors. Small liver sections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality. Sanger sequencing and the NanoString nCounter® technology were used to assess the suitability of these samples in downstream molecular techniques. The results revealed that of the formalin fixatives, NBF samples fixed for 24 and 48 hr were superior to ZF samples for both Sanger sequencing and the Nanostring nCounter®. The non-formalin PAXgene® samples were equally successful and they showed greater stability in nucleic acid quality and concentration over longer fixation times. This study demonstrated that small quantities of preserved tissue from smallmouth bass can be utilized in downstream molecular techniques; however, future studies will need to optimize the methods presented here for different tissue types, fish species, and pathological conditions.

Larvicidal Activity and Histopathological Effect of Averrhoa bilimbi Fruit Extract on Aedes aegypti from Surabaya, Indonesia.

Averrhoa bilimbi has been long thought to have biological activity. The aim of this study was to determine the activity of primary and secondary metabolites from A. bilimbi fruit extract on Aedes aegypti larvae mortality and midgut histopathology. Experiment was performed to third-instar Ae. aegypti larvae collected from Surabaya, which then exposed to A. bilimbi crude fruit extract at various concentration for 24 hours. After exposure, larvae were evaluated of its mortality and fixed in 2.5% neutral buffer formalin before processed and sectioned into histological slides and stained with hematoxylin-eosin (HE). Statistical analysis was performed using Spearman rank correlation to determine histopathological damage on midgut of Ae. aegypti larvae. Phytochemical screening of A. bilimbi crude fruit extract found that it contained saponins, tannins, and terpenoids. Minimum concentration able to induce mortality on Ae. aegypti larvae (LC50) was 977 ppm, while LC90 was at 1380 ppm. Severe alteration of larvae midgut was found after 24 hours exposure to 2000 ppm extract. Features of damage mostly found in larvae midgut were disruption of the microvilli, columnar cell vacuolization, epithelial nucleus crossed midgut lumen, and basal membrane damage. Damage caused by fruit extract in midgut of Ae. aegypti third instar larvae inhibited development of larvae. This study reported first finding of histopathological effect of A. bilimbi fruits extract on Ae. aegypti larvae midgut. Result of study was expected to contribute to better understand extract bioactivity of this plant to be applied as natural larvicide for Ae. aegypti.

Trichosanthes dioica Roxb. prevents hepatic inflammation and fibrosis in CCl4-induced ovariectomized rats

Summary Plant-derived natural products have shown beneficial effects in many diseases associated with liver. The present study aimed to appraise the effects of Trichosanthes dioica (TD) peel extract on oxidative stress, inflammation as well as fibrosis exhibited by the ovariectomized rats in which hepatic damage had been induced by the administration of carbon tetrachloride (CCl4). The CCl4 administration in the ovariectomized rats elevated the plasma ALT, AST, and ALP level in comparison with the control while TD peel extract showed significant reduction of those activities. Besides, oxidative stress and lipid peroxidation also increased in CCl4-treated rats. TD peel extract significantly reduced the oxidative stress marker's concentration and improved the catalase and SOD activities in CCl4-administered ovariectomized rats. These results suggest that TD peel extract is capable of protecting the liver from CCl4induced damage through preventing oxidative stress, lipid peroxidation, inflammation, fibrosis and boosting the diminished cellular antioxidant defense.

Comparative Micrometrical Studies on the Lungs of Pashmina, Bakerwali and Non-descript Goats of the Union Territories of Jammu and Kashmir and Ladakh

Background: The diversity of different climatic conditions and altitudes of UTs of Jammu and Kashmir and Ladakh (erstwhile J and K state) has given rise to differential adaptive mechanisms that enable goats to cope effectively with a variety of stressful environmental conditions. Jammu city (with an altitude of 327m to 412 m), like the rest of north-western India, features a humid subtropical climate. Ladakh is the dry temperate region and the highest plateau in the state of Jammu and Kashmir with much of it ranging from 3,000 m (9,800 ft) to 5,000–5,500 m (16,000–18,050 ft). There has been no systemic study on the probable comparative variation in micrometry of lungs in high altitude Pashmina goats of Ladakh and migratory Bakerwali and non-descript goats of Jammu and Kashmir, which live in regions with varied altitudes. Respiratory system plays a vital role in exchange of oxygen, olfaction, phonation and thermoregulation of the body. No literature on the comparative micrometry of the lungs in these three goat populations was found and such paucity of available literature prompted this present study. Methods: The work was done in the lung samples of adult Pashmina, Bakerwali and non-descript goats (N=10 each) collected from slaughter houses of UT of Ladakh and in and around Jammu city of UT of J and K. The representative tissue samples from lungs of all the goats were preserved in 10% Neutral Buffered Formalin (NBF) solution, processed for paraffin block preparation, tissue sections of 5 mm thickness were obtained and stained with Hematoxylin and Eosin to record various micrometrical parameters. Results: Our study revealed that significantly largest alveolar diameter and thinnest inter alveolar septal thickness was recorded in Pashmina goats followed by Bakerwali and non-descript goats. This might be due to the adaptive morphological phenomenon of the Pashmina goats which are natural habitants of high altitudes of the Himalayan regions for better utilization of the oxygen in hypoxic conditions.

Effects of Pesticides-Laden Fruits and Vegetables on the Permeability of Renal Basement Membranes-A Route to Proteinuria

&#x0D; &#x0D; &#x0D; Objective: To study the hazardous effects of pesticide residue-laden fruits and vegetables on the permeability of renal basement membrane at proximal and distal convoluted tubules in rats.Study Design: Observational study. Place and Duration of Study: The study was conducted at the animal house at the Postgraduate Medical Institute (PGMI), Lahore for 21 consecutive days from 15th Nov 2018 to 6th Dec 2018. Materials and Methods: The study design was comparative comprising of twelve Wistar male rats. The subjects were divided into two equal groups, control (group A) and experimental (group B). Body weight of subjects were noted before and after the experiment. Triazophos was given to the group B in a dose of 8.2 mg/kg body weight-sub chronic dose (1/10th of LD50) as 1:100 dilution solution by oral gavage for 21 consecutive days. Blood for serum creatinine and 24-hour urine for proteinuria were collected on day 1, 7, 14 and 21. On day 22, the subjects were sacrificed, kidneys dissected out and preserved in 10% neutral buffered formalin (NBF) for histological examination. Pre and post experiment body weight of control group was compared with experimental group. Results: The histopathology of the renal basement membrane of group A showed continuity while group B showed interruption, vacuolization, inflammatory cell infiltration and blood vessel congestion in tissues. Biochemical analysis confirmed raised serum creatinine and proteinuria levels. Conclusion: The pesticide residue-laden fruits and vegetables adversely affect the histology of basement membranes in proximal and distal convoluted tubules leading to disturbed renal functions. The presence of residual pesticides in the food chain is hazardous to human health.&#x0D; &#x0D; &#x0D;

Localization and immunohistochemical detection of swine influenza A virus subtype H1N1 antigen in formalin-fixed, paraffin-embedded lung tissues from naturally infected pigs

BackgroundSwine influenza A viruses (SIV) infection is among the leading causes of respiratory diseases in a number of animal species and human, and has been reported to cause substantial losses to pig industry. Previous reports of serological, molecular, and surveillance studies in commercial piggeries in Nigeria indicated the presence of SIV subtypes H1N1 and H3N2 in infected pigs; hitherto, there exists lack of studies on the pulmonary pathology and pathogenicity of SIV in Nigeria. This study investigates the presence of SIV subtype H1N1 antigen in the formalin-fixed paraffin-embedded lung sections obtained from apparently healthy pigs slaughtered at abattoirs located in Lagos, Ogun, and Oyo States, Southwest Nigeria using a streptavidin-biotin (ABC) immunoperoxidase (IP) staining. Two hundred four lungs consisting of 144 grossly pneumonic lungs and 60 apparently normal lungs were randomly collected, fixed in 10% neutral-buffered formalin, embedded in paraffin wax, and processed for histopathological examination and immunohistochemistry.ResultsThe main gross lesions were marked pulmonary edema and mild bilateral consolidation of cranial lobes. Histopathology revealed suppurative bronchitis, and bronchiolitis with or without concurrent widespread degeneration and necrosis of epithelial cells (52.08%) and thickening of alveolar septa due to cellular infiltration consisting predominantly of neutrophils and mononuclear cells (macrophages and plasma cells) (39.58%). The lumina of most airways contained exudate consisting of neutrophils, desquamated epithelia cells, and necrotic debris. SIV antigen was immunohistochemically detected in 7/204 (3.43%) samples using SIV-specific (H1N1) monoclonal antibody. Positive cells exhibited a typical dark-brown reaction in the infected cells. A strong positive immunohistochemical staining was detected mainly in the alveolar macrophages and bronchial submucosal glandular epithelial cells while less intense staining was observed in the bronchiolar epithelial cells.ConclusionsThe present study describes the distribution and localization of SIV subtype H1N1 antigens in the lung tissues of the infected pigs and provides public awareness on the presence of the virus in pig population in Nigeria and the risk factors associated with the infection. Therefore, people working in pig farms should maintain high level of biosafety and personal hygiene. This is the first report of immunohistochemical detection of SIV subtype H1N1 antigen in naturally infected pigs in Nigeria and may indicate rapid dissemination of the virus in susceptible pigs in the study area. A further molecular epidemiological study to investigate other SIV subtypes circulating in Nigerian pig population is warranted.

A Tale of Three Fixatives: Detection of Middle East Respiratory Syndrome Coronavirus in Paraffin‐Embedded Tissues Preserved in Three Fixatives

Middle East respiratory syndrome coronavirus (MERS‐CoV) is a zoonotic pathogen endemic to dromedary camels known to cause a respiratory illness in humans that is fatal in 35% of cases. As such, inactivated tissues provide a safer format when testing for this virus. Neutral buffered formalin (NBF), pathology’s gold standard for tissue fixation, preserves histomorphology and inactivates many pathogens. However, NBF causes the crosslinking and fragmentation of proteins and nucleic acids in tissues. This results in a rapid decline in biomolecular quality after just 24 hours and damage increases thereafter. NBF is also a volatile organic compound and carcinogen. Two novel fixatives, phosphate‐buffered 70% ethanol (BE70) and BE70 with added guanidium salts (BE70G), are alternative preservatives that cause less damage to biomolecules, even in tissues immersed for longer than 3 months. These ethanol (EtOH)‐based fixatives have equivalent histomorphology and inactivation profiles as well as better human safety profiles. We hypothesized that existing anti‐MERS‐CoV immunohistochemistry (IHC) and in situ hybridization (ISH) protocols would work on tissues preserved with these EtOH‐based fixatives.Roughly equivalent tracheal and nasal turbinate samples from eight alpacas experimentally infected with MERS‐CoV were fixed in 10% NBF, BE70, and BE70G, processed into paraffin and sectioned for hematoxylin and eosin staining or immunolabeling. Anti‐MERS‐CoV nucleocapsid IHC using polymer‐based detection and MERS‐CoV specific ISH using RNAscope® 2.5 HD brown detection were conducted on all samples. Because many EtOH‐based fixatives do not require antigen retrieval (AR), ISH and IHC were run with and without AR on the EtOH‐based fixed tissues.Although MERS‐CoV IHC and ISH appropriately labeled target molecules in all samples, signal strength varied across the fixatives. The strongest labeling was seen in NBF, followed by BE70G, and finally BE70. This result was not unexpected as the protocols were optimized for use with NBF tissues. Interestingly, both BE70 and BE70G required AR to successfully visualize the targets by both IHC and ISH, although BE70G did have weak ISH signal without AR. Histomorphology for all three fixatives without AR was very similar. However, AR blurred cell borders and reduced overall cellular definition in the EtOH‐based fixed samples, particularly BE70G.Taken together, these results support the use of these novel EtOH‐based fixatives as alternatives to NBF, particularly when biomolecule preservation for downstream molecular biology assays is critical. Finally, BE70 and BE70G increase human safety by inactivating pathogens with less harmful chemicals as well as provide a more flexible fixation time frame. This is particularly useful for high containment work as it would allow safe transfer of inactivated tissues to lower biosafety level labs while better preserving the samples for downstream analysis.Support or Funding InformationThis work was funded by the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University and the USDA APHIS NBAF Scientist Training Program.Histomorphology, Immunohistochemistry (IHC), and In Situ Hybridization (ISH) of NBF‐, BE70‐, and BE70G‐Fixed Tissues Column 1 is hematoxylin and eosin staining, Column 2 is anti‐MERS‐CoV nucleocapsid IHC, and Column 3 is MERS‐CoV‐specific ISH in fixed, paraffin‐embedded nasal turbinates. All immunolabeled tissues were counterstained with hematoxylin. Row 1 is NBF‐fixed tissue. Row 2 is BE70‐fixed tissue. Row 3 is BE70G‐fixed tissue. The bar for all main images is 100 microns.Figure 1

MicroCT Scanner to Differentiate Histotechnological Processing of Bone Tissue Using Different Nucleic Acid‐Based Fixatives

Micro‐computed tomography (μ‐CT) scanners permit researchers to characterize the three‐dimensional structure of bone. They allow for the visualization of many bone structural parameters to be quantified with an extremely high degree of precision. Immunohistochemistry (IHC) (colorimetric) and immunofluorescence (IF) allows for characterization of DNA and RNA in fixed tissue sections. μ‐CT scanners along with IHC and IF allows processed tissue sections to be examined by a variety of different fixatives, in order to characterize the effects of fixation on bone morphology (i.e., adult chicken and mice), and preservation of different types of intact, non‐denatured nucleic acids (e.g., right‐handed B‐DNA, left‐handed Z‐DNA). Fixation was performed using 10% Neutral Buffered Formalin (NBF), Formalin‐Alcohol‐Acetic Acid (FAA), Davidson’s fixative, and Carnoy’s solution for 72 hours at room temperature. Both decalcified and non‐decalcified bone tissues were processed. Ultra‐pure molecular biological grade fixatives and water were used in order to ensure the best histotechnological data. Tissues processed in FAA resulted in hardening of tissue, but good fixation of intact DNA. Tissues processed in 10% NBF resulted in softer tissue, but poor fixation of intact DNA and required antigen retrieval (AR). Tissues processed in Carnoy’s solution resulted in hardened tissue, but excellent DNA IHC and IF. Tissues processed in Davidson’s fixative resulted in hardened tissues, but good preservation of DNA. We will try to correlate structural bone differences observed with the μCT scanner, relative to DNA isolation and IHC and IF staining of bone tissue sections with anti‐B‐DNA and anti‐Z‐DNA monoclonal antibodies. These results will better enable researchers to histotechnologically process tissue while obtaining data on DNA structure and function.

Evaluation the effect of using thyme and oregano powder in comparison to the antibiotic and probiotic supplementation on growth, some immune responses and intestinal morphology of broiler chicks

To determine the effect of using thyme and oregano powder supplementation on performance, some immune response and intestinal morphology of broiler chicks a total of 200 one-day-old Cobb 500 male broiler chicks were divided into the four treatments consisting of five replicates of 10 chicks each. The treatments were as control groups with no herbal, antibiotic and probiotic supplementations. control groups + virginiamycine antibiotic, control groups + protexin probiotic, control groups + 2 % of each thyme and oregano powder supplementation and Control groups + 4 % of each thyme and oregano powder supplementation. The performance parameters such as feed intake, body weight gain and feed conversion ratio were measured during the study period. To determine immunity parameters on days 28, 0.5 mL of 10 % sheep red blood cells suspension was administrated to two chicks per each replicate through and at day 31, 3 mL blood samples were collected from left wing. On day 42, jejunum tissue samples were separated and fixed in (10 %) neutral buffer formalin and were analyzed by auto-processing apparatus, there after the slide sections were prepared by rotary microtome type (Manubeni, Erma-Tokyo, Japan) and the average of villi measurements was reported as a mean for each bird. The result of this study showed that the dietary inclusion of herbals and probiotic had no significant effects on FI and FCR of broiler chicks but higher body weight gain were seen with they received thyme+oregano supplementation. Inclusion of thyme+oregano and probiotic increased humeral immunity in broiler chicks compared to the control (P ≤ 0.05). Additionally heterophil, lymphocyte and their ratio were not influenced by experimental treatments. The villus length, villus width, and crypt depth were increased were the birds feed by oregano and thyme powders. In conclusion in current study some of beneficial acts toward using thyme and oregano powders on performance, some immune parameters and intestinal morphology on broiler chicks were demonstrated.

Morphological features of pulmonary fibrosis in workers occupationally exposed to alpha radiation

Purpose: The article reports on a comparative analysis of biological specimens of lung tissues collected from workers with pulmonary fibrosis induced by internal exposure to plutonium alpha-particles (plutonium-induced pulmonary fibrosis [PuPF]) and with etiologically different pulmonary fibrosis (non-PuPF) that developed as an outcome of a chronic obstructive pulmonary disease (COPD).Materials and methods: To perform histological examinations, lung tissues were sampled during autopsy. Six samples of various lung regions (the apical region, the lingula of the left lung and the inferior lobe) were collected from each donor. The resected tissue samples were fixed in 10% neutral-buffered formalin during 24 h and embedded into paraffin blocks (FFPE). FFPE blocks with lung tissue specimens collected from 56 workers with PuPF, 34 workers with non-PuPF and 35 workers without any lung disease were used in the study. To perform microscopic examination, lung tissue specimens were hematoxylin and eosin stained. To examine the connective-tissue scaffold of lung stroma and identify foci of pulmonary fibrosis, the cut sections of paraffin blocks were stained by Van Gizon’s method (to assess the total volume of fibrosis-affected tissues), Gomori’s technique (to define the reticular scaffold of lung stroma) and Weigert’s technique (to examine elastic fibers). Morphological patterns of all biological specimens were studied using immunohistochemistry. To fit the empirical data, the Weibull’s model was used.Results and conclusions: The study found qualitative and quantitative morphological features specific for PuPF compared to non-PuPF. The study demonstrated that hyper-production of collagen type V plays a key role in PuPF. The collagen type V content in fibrotic foci in lung tissue specimens from workers with PuPF was found to be increased.

Use of Microct Scanner to Characterize the Histotechnological Processing of Bone using Different Tissue Fixatives: Relationship to DNA Preservation (Immunohistochemistry)

Micro-computed tomography (micro-CT) scanners allow for the three-dimensional analysis of bone structure to be visualized and numerous bone structural parameters to be quantified with a high degree of accuracy. Nucleic acid-based immunohistochemistry (IHC) allows for characterization of the presence, amounts and different types of DNA in paraffin-embedded fixed tissue sections. Micro-CT scanners (i.e., Bruker SkyScan 1173 µCT system), along with immunohistochemically processed tissue sections were used to examine a variety of different fixatives in order to characterize the effects of fixation on bone morphology (i.e., adult chicken), and preservation of different types of intact, non-denatured nucleic acids (e.g., B-DNA, Z-DNA). Several different types of bone (e.g., femur, tibia, and vertebra) were characterized. Fixation was performed using 10% Neutral Buffered Formalin (NBF), and Formalin-Alcohol-Acetic Acid (FAA) for 24, 48, 72 and 96 hours at room temperature. Both decalcified and non-decalcified bone tissues were processed. Molecular biological grade fixatives and water were used in order to ensure the best histotechnological results. Tissues processed in FAA resulted in hardening of tissue, but good fixation of intact B-DNA and Z-DNA. Tissues processed in 10% NBF resulted in softer tissue, but poor fixation of intact DNA. Tissues processed in 10% NBF required antigen retrieval. Our group is correlating morphological differences observed with different fixatives using a microCT scanner, relative to DNA identification employing immunohistochemical staining of bone tissue sections with anti-B-DNA and anti-Z-DNA monoclonal antibodies. Our data demonstrates that FAA results in easier histotechnological processing of bone tissue, better morphological results and superior IHC staining. Research project was supported by an NSF grant, Award ID No: 1828305. .