Academic literature on the topic 'Standard Stained Shingle Co'

Objective: To compare the bichemical and morphological effects of L -Arginine against the changes caused by butter and corn oil supplementation Study design: A prospective experimental study Place: Department of Anatomy BMSI, JPMC Duration: August to October 2008. Methodology:Male Albino rats weighing 200 - 240gm were selected and divided into 5 groups. Group ‘CL’ received standard laboratory diet. Group ‘Bu’ received 20% added unsalted butter in diet. Group ‘Co’ received 20% added corn oil in diet. Group ‘BuAr’ received 20% Butter with L-Arginine 300mg /kg body weight /day orally .Group‘CoAr’ received 20%corn oil along with L-Arginine 300mg/kg body weight/day orally. On completion of study period that is 4 weeks, animals were sacrifised. Blood was drawn for hormonal assays. Adrenal glands were removed and fixed in buffered neutral formalin. Right adrenals were processed and sectioned at4 µm thickness to be stained with Mallory trichrome stain to visualize blood vessel. Left adrenalswere sectioned with cryostat in 10µm sections and stained with Oil red O to visualize fat in cells. Results:Highly significant and moderately significant decrease observed in ACTH (Adrenocorticotrophic hormone)levels in Group BuArand CoAr when compared to Bu and Co respectively; insignificant difference was found between BuAr&CoAr. Moderately significant and significant decrease observed in corticosterone levels in Group BuAr and CoAr when compared to Buand Co respectively. Insignificant difference was found between BuAr and CoAr . Mallory trichrome stained section showed less dilated blood vessels in BuAr&CoAr compared to Bu & Co respectively, while difference among the former two was not remarkable. Oil red O stained sections showed less densely packed fat globules in group BuAr&CoAr compared to Bu and Co respectively. Difference between BuAr&CoAr was not marked. Conclusion: Butter has more stimulatory effect on adrenal cortical cells but the comparison with corn oil is not statistically significant except for ACTH levels. L Arginine seems to be effective in lowering the levels of stress hormones, fat accumulation and vasodilatation when given along with corn oil and butter oil.

The identification of protein aggregates as biomarkers for neurodegeneration is an area of interest for disease diagnosis and treatment development. In this work, we present novel super luminescent conjugated polyelectrolyte molecules as ex vivo sensors for tau-paired helical filaments (PHFs) and amyloid-β (Aβ) plaques. We evaluated the use of two oligo-p-phenylene ethynylenes (OPEs), anionic OPE12- and cationic OPE24+, as stains for fibrillar protein pathology in brain sections of transgenic mouse (rTg4510) and rat (TgF344-AD) models of Alzheimer’s disease (AD) tauopathy, and post-mortem brain sections from human frontotemporal dementia (FTD). OPE12- displayed selectivity for PHFs in fluorimetry assays and strong staining of neurofibrillary tangles (NFTs) in mouse and human brain tissue sections, while OPE24+ stained both NFTs and Aβ plaques. Both OPEs stained the brain sections with limited background or non-specific staining. This novel family of sensors outperformed the gold-standard dye Thioflavin T in sensing capacities and co-stained with conventional phosphorylated tau (AT180) and Aβ (4G8) antibodies. As the OPEs readily bind protein amyloids in vitro and ex vivo, they are selective and rapid tools for identifying proteopathic inclusions relevant to AD. Such OPEs can be useful in understanding pathogenesis and in creating in vivo diagnostically relevant detection tools for neurodegenerative diseases.

Quantification of parasitemia is an important part of a microscopic malaria diagnosis. Giemsa-stained thin blood smear is the gold standard method for detecting malaria parasite enumeration. However, manual counting reveals the limitations of human inconsistency and fatigue, as well as the unreliability of accuracy and non-reproducibility. Inaccurate parasitemia affects clinical diagnosis and therapeutic procedure. Automated quantification is therefore useful to improve the performance of quantifying parasite density. In this paper, the texture-based classification approach is investigated. The methods consist of the following processes: pre-processing, segmentation, feature extraction and the classification of erythrocytes. The pre-processing is applied for image conversion and enhancement. The segmentation combines local adaptive thresholding, morphological process and watershed transform to extract red blood cells, separate touching and overlapping cells. Texture analysis is performed to establish parameters obtained from first-order, second-order and higher-order statistical analysis and wavelet transform. Two feature selection approaches, the sequential forward selection method and sequential backward elimination method, integrated with a support vector machine classifier are examined to obtain the optimal feature set for identifying the Plasmodium falciparum stages. We found that gray-level co-occurrence matrices based textural features were highly selected. The optimal feature set selected by the sequential forward selection yields lesser number of features and tends to give a higher degree of accuracy than the feature set selected by sequential backward elimination. The proposed method produces 98.87% accuracy for binary classification, 99.56% accuracy for ring stage classification, and 99.48% accuracy for tropozoite stage classification. Grey-level co-occurrence matrices based texture analysis is the dominant method compared to first-order and higher-order statistical texture analysis as well as wavelet transform.

Abstract Background: Hematoxylin and eosin staining (H&E) is widely used as an anatomical assay for clinical diagnosis. Researchers and clinicians also rely on molecular in situ techniques, such as multiplexed immunofluorescence (mIF), to gain deeper insights on cellular phenotypes and tissue microenvironment. As a result, there has been significant interest in combining anatomical stains with molecular imaging techniques, most commonly by using a terminal H&E stain after mIF staining. This allows a combination of the two assays but has been observed to show alterations in the H&E staining pattern. In this work we identify heat-induced epitope retrieval (HIER) as the root cause of alterations of the terminal H&E stain after mIF. We further demonstrate a new workflow combining an initial H&E stain with subsequent InSituPlex assay, to avoid these alterations and thus enable co-registration of highly sensitive multiplexed immunofluorescence with an unaltered H&E stain. Methods: FFPE tissue slides were stained using a standard HIER protocol or a full mIF assay, followed by a standard H&E stain. Slides were imaged using a Zeiss Axioscan.Z1 scanner. Additional H&E-stained slides were prepared, and the H&E stain removed using a destaining protocol before carrying out InSituPlex® mIF staining for multiple markers. Fluorescence and brightfield images of the same slide were overlaid using UltiStacker® to assess qualitative differences between pre- and post-mIF H&E, and UltiAnalyzer.AI® was used to quantify cell densities and signal intensities between mIF image pre- and post-H&E. Results: Alterations in H&E staining were observed between slides directly stained with H&E and slides stained with a terminal H&E after HIER alone or a full mIF assay. Calculated cell counts and tissue area were consistent throughout, but some tissue microstructures could be difficult to resolve after either HIER or full mIF. Our destaining protocol was successful in removing hematoxylin and eosin from directly-stained H&E slides and allowing subsequent mIF staining. For most biomarkers, qualitative and quantitative differences in InSituPlex mIF staining were minimal. Brightfield and fluorescent images were co-registered with sub-micron accuracy using UltiStacker, enabling molecularly defined cellular phenotyping within a traditional H&E image. Conclusions: While the mIF to H&E workflow is widely used and is capable of allowing combined anatomical and cellular phenotyping, alterations in the H&E stain exist due to the use of HIER in most mIF protocols. An alternative method is possible in which H&E-stained slides are destained and then restained for mIF or other spatial assays, providing the same valuable combination of assay information but without the HIER-induced alterations in H&E staining pattern. Citation Format: Kevin Hwang, Grace Vezeau, Edyta Olejnik, Douglas Wood, Ruben Cardenes, Lauren Duro, Gourab Chatterjee, Je H. Lee. A novel method to minimize HIER-induced alterations on H&E staining in an integrated mIF-H&E workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3767.

Abstract PR1 is an HLA-A2-retricted, nonameric peptide that is derived from the azurophil granule proteases neutrophil elastase (NE) and proteinase 3 (P3). PR1 has been targeted successfully in acute (AML) and chronic (CML) myeloid leukemia using anti-PR1/HLA-A2 antibody (8F4), PR1-peptide vaccine and PR1-specific cytotoxic T lymphocytes (PR1-CTL). We have previously reported that NE and P3 are cross-presented by normal B cells and dendritic cells (DC), leading to PR1 expression by HLA-A2. Since multiple myeloma (MM) is a B cell malignancy, we investigated whether MM cells can cross-present PR1 as a possible target for immunotherapy. To study whether PR1 is presented by MM cells, patient bone marrow (BM) was stained with 8F4 antibody and then imaged using confocal microscopy. PR1/HLA-A2 was detected on the surface of CD138+ MM cells in BM samples from 3 of 6 HLA-A2+ MM patients (Fig. 1). We then investigated whether cellular immunity to PR1 is detected in peripheral blood (PB) from patients with MM. PB samples from MM patients who had undergone allogeneic (allo) (n=9) and autologous (auto) (n=2) stem cell transplantation (SCT) were stained with PR1/HLA-A2 dextramer in addition to standard lineage markers. PR1-specific CD8+CTL were detected in PB of 10 of 11 patients (range=0.02%-2.9%). Because P3 and NE expression is limited to myeloid cells, we sought to determine the mechanism of PR1 presentation in MM. We performed RT-PCR and western blotting on seven MM cell lines, including U266, ARK, ARP-1, OPM-2, LP-1, IM-9 and RPMI 8226. Neither NE nor P3 were detected in the MM cell lines studied at either the mRNA or proteins levels. We then investigated whether MM cells took up NE and P3. We cultured MM cells for 30 hours with 10 ug/mL of soluble NE and P3 or irradiated HLA-A2 negative PMN, the latter as a source for cell-associated NE and P3. Cells were then stained intracellularly for NE and P3 at different time points. Flow cytometry analysis showed that all the cell lines analyzed took up NE and P3. Uptake was seen as early as 1 hour after co-culture with soluble NE and P3 and was higher for soluble P3. Additionally, more uptake was seen in the cells that were co-cultured with irradiated PMN in comparison with soluble NE and P3. We then investigated whether PR1 expression in MM was through NE and P3 cross-presentation. We focused our studies on the HLA-A2+ U266 MM cell line. U266 cells were co-cultured with soluble NE, P3 or irradiated PMN, as described in the previous section, and then surface stained with 8F4. We detected PR1/HLA-A2 on the surface of U266 cells by flow cytometry as early as 6 and 24 hours after co-culture with soluble NE and P3, respectively. Cross-presentation was also seen in the cells that were co-cultured with irradiated PMN to a similar extent in comparison with the cells that were cultured with soluble NE and P3, however, cross-presentation occurred at an earlier time point (1 hour) in the cells that were cultured with PMNs. Cross-presentation was abrogated by lactacystin, a proteasome inhibitor, and brefeldin A, an ER/Golgi transport inhibitor, indicating that NE and P3 cross-presentation occurs through conventional cross-presentation mechanisms. Furthermore, because immune modulatory drugs and proteasome inhibitors are part of the standard of care therapy for MM, and since both have been shown to affect cross-presentation by DC, we tested whether lenalidomide and bortezomib altered PR1 cross-presentation by U266 and normal DC. U266 and DC were cultured with irradiated PMN in the presence of increasing doses of bortezomib or lenalidomide and then stained with PR1/HLA-A2. Flow-cytometry analysis showed a significant inhibition of PR1-cross-presentation by U266 after addition of bortezomib, but not lenalidomide. PR1 cross-presentation by DC was not affected by either drug. Finally, we tested whether PR1 cross-presentation caused MM cells to become susceptible to PR1-targeting immunotherapies. U266 cells were cultured with NE or P3 for 24 hours, the time point that showed the maximal cross-presentation, followed by addition of 8F4 antibody or co-culture with PR1-CTL. Using calcein AM cytotoxicity assays, we showed dose dependent killing of U266 by 8F4 and PR1-CTL following PR1 cross-presentation (Fig. 2). Together our data show that PR1 is cross-presented by primary MM cells and cell lines. These findings lay the foundation for the future applications of PR1-targeting immunotherapies in MM. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

It is known that multipotent stromal cells (MSCs) and thymocytes possess membrane affinity and interaction in the thymic niches that is essentially important for thymocytes differentiation. However there are no data about possible influence of intercellular contacts in the reverse direction: from the thymocytes to the MSCs.Materials and methods. The MSCs were obtained from the thymuses of С57ВL mice, using the explants technique, and cultivated under standard conditions during 8-12 passages. Thymocytes or bone marrow cells (106) were added to 4×104 MSCs for 24 hours. Thereafter they were eliminated and standard culture medium was changed by osteogenic or adipogenic differentiation medium and cultured during 10 days. After fixation the cells were stained by 1 % alizarin red S solution or 0.2 % solution of oil red О respectively. After extraction of the stains with 10 % acetic acid or isopropyl alcohol the optic density of extracts at 520 nm was measured.Results. We found that thymic multipotent stromal cells of the C57BL mice were effectively differentiated in vitro into the osteogenic and adipogenic lineages in the appropriate differentiation media that was evidenced by alizarin red and oil red staining of cell cultures. According to the results of measurement of optic density of the dye extracts, it was found that effectiveness of thymic MSCs differentiation into the osteogenic lineage after prior short-term co-cultivation with the thymocytes is increased.Conclusions. The contact of thymic stromal cells with thymocytes but not with bone marrow cells in the previous 24 hours potentiates the osteogenic differentiation and has no effect on the adipogenic cells maturation.

Traumatic brain injury (TBI) is a chronic, life threatening injury for which few effective interventions are available. Evidence in animal models suggests un-checked immune activation may contribute to the pathophysiology. Changes in regional density of active brain microglia can be quantified in vivo with positron emission topography (PET) with the relatively selective radiotracer, peripheral benzodiazepine receptor 28 (11 C-PBR28). Phenotypic assessment (activated vs resting) can subsequently be assessed (ex vivo) using morphological techniques. To elucidate the mechanistic contribution of immune cells in due to TBI, we employed a hybrid approach involving both in vivo (11 C-PBR28 PET) and ex vivo (morphology) to elucidate the role of immune cells in a controlled cortical impact (CCI), a rodent model for TBI. Density of activated brain microglia/macrophages was quantified 120 hours after injury using the standardized uptake value (SUV) approach. Ex vivo morphological analysis from specific brain regions using IBA-1 antibodies differentiated ramified (resting) from amoeboid (activated) immune cells. Additional immunostaining of PBRs facilitated co-localization of PBRs with IBA-1 staining to further validate PET data. Injured animals displayed greater PBR28suv when compared to sham animals. Immunohistochemistry demonstrated elevated density of amoeboid microglia/macrophages in the ipsilateral dentate gyrus, corpus callosum, thalami and injury penumbra of injured animals compared to sham animals. PBR co-stained with amoeboid microglia/macrophages in the injury penumbra and not with astrocytes. These data suggest the technologies evaluated may serve as bio-signatures of neuroinflammation following severe brain injury in small animals, potentially enabling in vivo tracking of neuroinflammation following TBI and cellular-based therapies.

The newly synthesized compounds are being tested for in vitro anticancer activity. The method used for In Vitro testing is Sulforhodamine B assay also known SRB assay. Cell lines were prepared and homogenized and disassociated with the help of trypsin. Then trypsin was inactivated with fetal bovine serum. Then cell concentration was determined. Synthesized molecules were prepared into four different dilutions and exposed to cell lines. The procedure was also compared with standard drug doxorubicin. All the cell medium were incubated 37 degrees centigrade in a humidified incubator with 5 percentage CO. The plates were stained and fixed with trichloroacetic acid. Finally, the plates were incubated in orbital shaker incubator and absorbance was measured in a microplate reader at 510nm. All compounds (1-30) showed the similar anticancer activity of compounds (IA, IB, ID, IE, IF, IIB, IIC, IIIA, IVB, IVF, VA, VC, VD, VE.) were more potent when compared to the rest of the compounds synthesized.

AbstractThis aim of this study was to assess the discriminatory value of fractal and grey level co-occurrence matrix (GLCM) analysis methods in standard microscopy analysis of two histologically similar brain white mass regions that have different nerve fiber orientation. A total of 160 digital micrographs of thionine-stained rat brain white mass were acquired using a Pro-MicroScan DEM-200 instrument. Eighty micrographs from the anterior corpus callosum and eighty from the anterior cingulum areas of the brain were analyzed. The micrographs were evaluated using the National Institutes of Health ImageJ software and its plugins. For each micrograph, seven parameters were calculated: angular second moment, inverse difference moment, GLCM contrast, GLCM correlation, GLCM variance, fractal dimension, and lacunarity. Using the Receiver operating characteristic analysis, the highest discriminatory value was determined for inverse difference moment (IDM) (area under the receiver operating characteristic (ROC) curve equaled 0.925, and for the criterion IDM≤0.610 the sensitivity and specificity were 82.5 and 87.5%, respectively). Most of the other parameters also showed good sensitivity and specificity. The results indicate that GLCM and fractal analysis methods, when applied together in brain histology analysis, are highly capable of discriminating white mass structures that have different axonal orientation.

We developed a method for detecting activity of axonal cholinesterase (CE) and carbonic anhydrase (CA)--markers for motor and sensory nerve fibers (NFs)--in the same histological section. To reach this goal, cross-sections of muscle nerves were sequentially incubated with the standard protocols for CE and CA histochemistry. A modified incubation medium was used for CA in which Co++ is replaced by Ni++. This avoids interference of the two histochemical reactions because Co++ binds unspecifically to the brown copper-ferroferricyanide complex representing CE activity, whereas Ni++ does not. Cross-sections of the trapezius muscle nerve containing efferent and afferent NFs in segregated fascicles showed that CE activity was confined to motor NFs. Axonal CA was detected solely in sensory NFs. The number of labeled motor and sensory NFs determined in serial cross-sections stained with either the new or the conventional technique was not significantly different. Morphometric analysis revealed that small unreactive NFs (diameter less than 5 microns) are afferent, medium-sized ones (5 microns less than d less than 7 microns) are unclassifiable, and large ones (d greater than 7 microns) are efferent. The heterogenous CE activity of thick (alpha) motor NFs is linked to the type of their motor units. "Fast" motor units contain CE reactive NFs; "slow" ones have CE negative neurites.