Life After Death

2020 ◽  
Author(s):  
Kavita Gaur ◽  
Puja Sakhuja ◽  
Kaushik Majumdar ◽  
Divya Arora Thakral
Keyword(s):  
Staining Pattern ◽  
Performance Characteristics ◽  
Positive Staining ◽  
Quality Maintenance ◽  
Life After Death ◽  
Staining Result ◽  
Background Staining

Abstract Objectives To test the performance characteristics of 69 primary immunohistochemistry antibodies after expiration and compare with fresh primary antibodies wherever possible. Methods A total of 69 expired primary antibodies were evaluated for specificity, background staining, and intensity. An optimal staining result corresponded to a semiquantitatively scored 2+ or 3+ intensity, with intact specificity devoid of moderate or strong background staining. Any deviation from a normal staining pattern was also considered to be a suboptimal result. Results Nearly half of the antibodies studied showed an optimally positive staining result after expiration (34/69, 49.2%). Overall, 10.1% (7/69) of antibodies could be compared with fresh primary antibodies of the same clone with equivalent results. Eight of 69 (11.6%) expired antibodies showed splotchy or granular staining. Conclusions Evidence from this study and previous work point to maintained functionality of a fair number of primary immunohistochemical antibodies after expiration. Decisions about the use of such reagents should be guided by a thorough assessment of functionality by the pathologist rather than a manufacturer-specified deadline. Quality maintenance should imply a sensible balance between histopathologic performance and economics.

scholarly journals COMPARATIVE STUDIES OF LIGHT MEROMYOSIN PARACRYSTALS DERIVED FROM RED, WHITE, AND CARDIAC MUSCLE MYOSINS

1971 ◽  
Vol 49 (3) ◽  
pp. 883-898 ◽  
Author(s):  
A. Nakamura ◽  
F. Sreter ◽  
J. Gergely
Keyword(s):  
Molecular Structure ◽  
Cardiac Muscle ◽  
Functional Properties ◽  
Comparative Studies ◽  
White Muscle ◽  
Staining Pattern ◽  
Uranyl Acetate ◽  
Positive Staining ◽  
Cardiac Muscles

Tryptic and chymotryptic light meromyosin paracrystals from red and cardiac muscles of rabbit show a negative and positive staining pattern with uranyl acetate and phosphotungstate that sharply differs from that of white muscle light meromyosin paracrystals. The main periodicity of about 430 A is the same regardless of the source of light meromyosin. The results are discussed in terms of the molecular structure and the functional properties of various myosins.

scholarly journals A Case of Silent Corticotroph Adenoma

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A563-A563
Author(s):  
Usonwanne U Ibekwe ◽  
Nathan Zwagerman ◽  
Srividya Kidambi ◽  
Jerald Peter Marifke
Keyword(s):  
Pituitary Adenoma ◽  
Cavernous Sinus ◽  
Endoscopic Resection ◽  
Staining Pattern ◽  
Positive Staining ◽  
Sellar Mass ◽  
Post Surgery ◽  
Morning Cortisol ◽  
Patient Reported ◽  
The Right

Abstract Background: Silent Corticotroph Adenomas (SCAs) are tumors with no biochemical or clinical features consistent with hypercortisolism but have positive immunostaining for ACTH. They account for approximately 1.1-6% of all pituitary adenomas and 5.5% of nonfunctioning adenomas. Clinical Case: A 49-year-old woman presented to clinic with a 6-month history of headache, vision changes, fatigue, hair thinning, brittle nails, lightheadedness, polydipsia, easy bruising, increased appetite, and weight gain of 178 pounds in 2 years. Labs obtained: morning cortisol 10 mcg/dl(7-25 mcg/dl), ACTH 59 pg/ml(7.2-63 pg/ml), IGF-1 96 ng/ml(52-328 ng/ml), LH 0.2 mIU/ml(1.9-12.5 mIU/ml), FSH 0.8 mIU/ml(2.5-10.2mIU/ml), alpha subunit <0.1 ng/ml(52-328 ng/ml), TSH 0.991 uIU/ml(0.358-3.74 uIU/ml), free T4 1.12 ng/dl(0.76-1.46 ng/dl), salivary cortisol 66 ng/dl(<100 ng/dl), 24-hour urine cortisol 10 mcg/24hr(3.5-45 mcg/24hr) and prolactin 64.3 ng/ml(2.8-29.2 ng/ml). No hook effect noted with serial dilution. MRI brain showed a 22 x 29 x 26 mm sellar mass extending into the suprasellar cistern displacing and compressing the optic nerves and chiasm superiorly with partial invasion into the right cavernous sinus. She had an endoscopic resection of the sellar mass. She developed diabetes insipidus post-operatively and required desmopressin transiently. In the immediate post-operative period, morning cortisol and ACTH were 16.9 ug/dl(6.2-19.4 ug/dL) 24.8 pg/ml(7.2-63.3 pg/ml) respectively. She was sent home without steroids. Pathology showed a staining pattern consistent with pituitary adenoma with positive staining for ACTH. One month after her surgery she was admitted with symptoms of orthostatic hypotension. Cortisol at 5pm was 3 ug/dl(2.3-11.9 ug/dl), ACTH 31.7 pg/ml(7.2-63.3pg/ml). She had a cosyntropin stimulation test done with peak cortisol of 19.3 ug/dl at 60 minutes. Due to her symptoms, she was started on oral hydrocortisone (HC) for secondary adrenal insufficiency (AI), but was eventually tapered off the steroids. Six months after her surgery, she developed worsening headaches. Repeat MRI obtained showed significant growth of the residual adenoma on the right side of the sella, invading the cavernous sinus. Morning cortisol level of 5.3 mcg/dl(4.3-22.4 mcg/dl) and ACTH level was 11 pg/ml(6-50 pg/ml). She had a repeat endoscopic resection of the pituitary tumor. Her post-surgery cortisol at 2 PM was 3 mcg/dl at which time patient reported symptoms of AI. She was discharged on HC. Pathology again showed a staining pattern consistent with pituitary adenoma with positive staining for ACTH. MIB-1 proliferative index was 5.6%. P53 immunostaining showed a moderate density of moderately intense nuclei in the adenoma. Conclusion: This case illustrates aggressive nature of SCAs with higher risk of recurrence compared to other non-functioning adenomas and therefore requires close follow up.

scholarly journals Occurrence and Antigenic Specificity of Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (P-ANCA) in Systemic Autoimmune Diseases

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2128
Author(s):  
Ourania D. Argyropoulou ◽  
Andreas V. Goules ◽  
Georgios Boutzios ◽  
Alexandra Tsirogianni ◽  
Charalampos Sfontouris ◽  
...  
Keyword(s):  
Autoimmune Diseases ◽  
Lupus Erythematosus ◽  
Staining Pattern ◽  
The Other ◽  
Cathepsin G ◽  
Antigenic Specificity ◽  
Positive Staining ◽  
Systemic Lupus ◽  
Systemic Autoimmune Diseases ◽  
Autoimmune Rheumatic Diseases

Perinuclear anti-neutrophilic cytoplasmic antibodies (P-ANCA) recognize heterogeneous antigens, including myeloperoxidase (MPO), lactoferrin, elastase, cathepsin-G and bactericidal/permeability-increasing protein. Although P-ANCA have diagnostic utility in vasculitides, they may also be found in patients with various other systemic autoimmune rheumatic diseases (SARDs). Nevertheless, the clinical significance and the targets recognized by P-ANCA in such patients remain unclear. For this purpose, herein we investigated the occurrence of ANCA-related antigenic specificities in 82 P-ANCA-positive sera by multiplex ELISA, as well as their association with other autoantibodies. The P-ANCA-positive sera corresponded to patients with vasculitides (n = 24), systemic lupus erythematosus (n = 28), antiphospholipid syndrome (n = 5), Sjögren’s syndrome (n = 7), rheumatoid arthritis (n = 3), systemic scleroderma (n = 1), sarcoidosis (n = 1) and Hashimoto′s thyroiditis (n = 13). In most P-ANCA-positive patients studied (51/82, 62.3%), these autoantibodies occurred in high titers (>1:160). The analysis of P-ANCA-positive sera revealed reactivity to MPO in only 50% of patients with vasculitides, whereas it was infrequent in the other disease groups studied. Reactivity to other P-ANCA-related autoantigens was also rarely detected. Our findings support that high P-ANCA titers occur in SARD. The P-ANCA-positive staining pattern is associated with MPO specificity in vasculitides, while in other autoimmune diseases, it mostly involves unknown autoantigens.

Performance Comparison of Commercial Fixing and Permeabilizing Reagents

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4927-4927
Author(s):  
Edward Joe ◽  
Tom Frey
Keyword(s):  
Light Scattering ◽  
Cell Surface ◽  
Performance Comparison ◽  
Positive Staining ◽  
Intracellular Staining ◽  
Cell Recovery ◽  
Cell Permeabilization ◽  
Ki 67 ◽  
Background Staining ◽  
Surface Staining

Abstract Flow cytometry is commonly used to characterize cellular antigens expressed both on the cell surface and inside the cell. As the technology advances, more researchers are using this tool to study intracellular activity to understand how cells communicate and respond to their environment. Intracellular targets of interest include cytoplasmic antigens and proteins, nuclear antigens and proteins, cytokines, etc. The quality of intracellular staining depends on the ability of the permeabilizing reagent to permeabilize the cell membrane for antibodies to get to the target of interest without destroying the cell. Five different commercial permeabilizing reagents were evaluated in a small study, Caltag Fix and Perm® Kit, BD IntraSure™ Kit*, BD FACS™ Permeabilizing Solution 2 (FACS Perm 2), 1X FACS Lysing Solution, and BD Cytofix/Cytoperm™ Kit. Five categories of performance were evaluated: cell recovery, maintenance of surface staining, preservation of light scattering properties, intracellular staining, and background staining. Cell recovery was assessed using CD45 APC and BD TruCOUNT™ tube; surface staining maintenance was assessed using CD45 APC, CD45 PerCP-Cy5.5, CD19 PE, and CD20 PerCP; intracellular and background staining were assessed using anti-bcl-2 FITC, anti-Zap 70 PE, anti-TdT FITC, anti-Ki-67 FITC, and CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5 reagents. Caltag and BD IntraSure Kits performed well in the category of cell recovery. Caltag Kit, BD IntraSure Kit, and FACS Lysing Solution maintained cell surface staining after cell permeabilization. BD IntraSure Kit and FACS Lysing Solution preserved light scattering properties of cells after permeabilization. Using signal-to-noise ratio as the criterion to evaluate background and intracellular positive staining, FACS Lysing Solution and FACS Perm 2 detected anti-bcl-2 FITC; BD IntraSure Kit and FACS Lysing Solution detected anti-TdT FITC; BD IntraSure Kit, FACS Lysing Solution, FACS Perm 2, and BD Cytofix/Cytoperm Kit detected anti-Ki-67 FITC; BD IntraSure Kit, FACS Lysing Solution, and FACS Perm 2 detected anti-Zap 70 PE; all kits detected CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5. Results showed that there is no universal permeabilizing reagent for all intracellular staining assays. Each test reagent has its own strength and weakness. Choosing the best permeabilizing reagent for a particular assay depends on the target of interest and the output of the assay.

scholarly journals Use of Monoclonal Antibodies To Identify Serotypes of Enterovirus Isolates

1998 ◽  
Vol 36 (7) ◽  
pp. 1877-1881 ◽  
Author(s):  
Alma S. Rigonan ◽  
Linda Mann ◽  
Tasnee Chonmaitree
Keyword(s):  
Monoclonal Antibodies ◽  
Preliminary Test ◽  
Laboratory Diagnosis ◽  
Neutralization Assay ◽  
Immunofluorescence Assay ◽  
Positive Staining ◽  
Type B ◽  
Staining Result ◽  
Cell Culture Isolation ◽  
Serotype Identification

Nonpoliovirus enteroviruses cause a variety of diseases that are common in young children and adults. The “gold standard” for laboratory diagnosis of enteroviruses is cell culture isolation, followed by serotype identification by neutralization assay. These procedures are time-consuming and expensive. Rapid serotype identification of enteroviruses is important in differentiating nonpoliovirus enterovirus pathogens from vaccine strain polioviruses that can be shed for some time after vaccination. In the present investigation, we evaluated a rapid method for serotype identification of enteroviruses by indirect immunofluorescence assay (IFA) using commercially available monoclonal antibodies for polioviruses, coxsackieviruses type B, and six serotypes of commonly circulating echoviruses. Of 291 isolates of enteroviruses included in the study, 95 were polioviruses and 196 were nonpoliovirus enteroviruses. Two hundred thirty-four of these (38 polioviruses and 196 nonpoliovirus enteroviruses) were consecutively grown in the laboratory over a 5-year period. IFA identified the serotypes of 74% of the consecutive isolates and 71% of all enterovirus isolates by yielding a positive staining result. The levels of agreement in the identification of the enterovirus group between IFA and neutralization tests were 92% for consecutively grown isolates and 85% for all enterovirus isolates. The sensitivity of the IFA for the detection of viruses for which specific monoclonal antibodies were applied was 73% for polioviruses, 85% for coxsackieviruses type B, and 94% for echoviruses. Specificity was near 100% for polioviruses and coxsackieviruses type B and 94% for echoviruses. We conclude that IFA can be helpful as a preliminary test for serotype identification of enteroviruses. The results are most accurate when the test identifies the isolate as a poliovirus.

scholarly journals Anti-Ovarian Vessels Antibodies in P. aeruginosa Rabbit Hyper Immune Sera, an Immunohistochemical Study

2017 ◽  
Vol 28 (1) ◽  
pp. 60
Author(s):  
Noor Al-Huda A. Saeed
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Endothelial Cell ◽  
Corpus Luteum ◽  
Blood Vessels ◽  
Immunohistochemical Study ◽  
Staining Pattern ◽  
The Other ◽  
Positive Staining ◽  
Rabbit Immunoglobulin ◽  
Tunica Adventitia

Cyclical angiogenesis in the ovary is a unique process supporting normal folliculogenesis as well as lute genesis. In this report we investigated the reactivity of rabbit anti pseudomonas aeruginosa antisera with ovarian blood vessels. Tissues stained with anti-sera were immunohistochemically visualized using biotinylated anti rabbit immunoglobulin and peroxidase conjugated streptavidin. Positive staining sites depend on anti-stain type, however, staining was observed in endothelial cell and tunica adventitia in most cases.On the other hand, corpus luteum blood vessels showed a positive staining pattern as well. We conclude from this study that a peculiar staining pattern was seen in ovarian blood vessels stained with rabbit anti-pseudomonas aeruginosa hyper immune sera, the importance of this reactivity need further investigation.

scholarly journals Light and electron microscopic localization of ACTH and pro-opiomelanocortin-derived peptides in human developmental and neoplastic cells.

1984 ◽  
Vol 32 (8) ◽  
pp. 885-893 ◽  
Author(s):  
R Y Osamura ◽  
Y Tsutsumi ◽  
K Watanabe
Keyword(s):  
Pituitary Adenomas ◽  
Staining Pattern ◽  
Secretory Granules ◽  
Small Cell ◽  
Electron Microscopic Level ◽  
Positive Staining ◽  
Electron Microscopic ◽  
Ectopic Acth ◽  
Pituitary Glands ◽  
Electron Microscopic Localization

Oncofetal aspects of ACTH and pro-opiomelanocortin (POMC)-derived peptides were studied immunohistochemically at the light and electron microscopic level in human fetal pituitary glands, pituitary adenomas, and small-cell carcinoma of the lung. ACTH, beta-endorphin, and gamma-MSH were localized in the same cells of both fetal and adult pituitary, as well as in the above-mentioned neoplastic tissues. However, alpha-MSH was observed only in the early fetal pituitary, its concentration decreasing with advancing gestational age. The adult pituitary contained only a few alpha-MSH-positive cells. By immunoelectron microscopy, ACTH in the adult pituitary was localized exclusively in the secretory granules. In fetal pituitary at 9 weeks' gestation, ACTH was localized in the perinuclear spaces (PNS), cisternae of rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules. The staining pattern of ACTH in these organelles varied from cell to cell. In fetal pituitaries of greater gestational ages, ACTH was localized in secretory granules. The pituitary adenomas mimicked the staining characteristics of the adult pituitary, i.e., negative or only very occasional alpha-MSH staining and localization of ACTH in the secretory granules. The ectopic ACTH-producing tumors showed a staining pattern similar to that of the early fetal pituitary, i.e., positive staining for alpha-MSH and the presence of ACTH in PNS and cisternae of RER.

scholarly journals Establishment of rostrocaudal polarity in tectal primordium: engrailed expression and subsequent tectal polarity

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1133-1144 ◽  
Author(s):  
N. Itasaki ◽  
H. Ichijo ◽  
C. Hama ◽  
T. Matsuno ◽  
H. Nakamura
Keyword(s):  
Staining Pattern ◽  
Mirror Image ◽  
Original Position ◽  
Positive Staining ◽  
Caudal Part ◽  
Retinotectal Projection ◽  
Projection Pattern ◽  
Rostrocaudal Axis ◽  
Positional Value ◽  
Tectal Polarity

In the E4 (embryonic day 4) chick tectal primordium, engrailed expression is strong at the caudal end and gradually weakens toward the rostral end. We used quail-chick chimeric tecta to investigate how the caudorostral gradient of engrailed expression is established and whether it is correlated with the subsequent rostrocaudal polarity of tectal development. To examine the positional value of the tectal primordium, we produced ectopic tecta in the diencephalon by transplanting a part of the mesencephalic alar plate heterotopically. In the ectopic tectum, the gradient of the engrailed expression reversed and the strength of the expression was dependent on the distance from the mes-diencephalon junction; the nearer the ectopic tectum was to the junction, the weaker the expression was. Consequently, the pattern of the engrailed expression in the host and ectopic tecta was nearly a mirror image, suggesting the existence of a repressive influence around the mes-diencephalon junction on the engrailed expression. We examined cytoarchitectonic development in the ectopic tecta, which normally proceeds in a gradient along the rostrocaudal axis; the rostral shows more advanced lamination than the caudal. In contrast, the caudal part of the ectopic tecta (near to the mes-diencephalon junction) showed more advanced lamination than the rostral. In both the host and ectopic tecta, advanced lamination was observed where the engrailed expression was repressed, and vice versa. Next we studied the correlation between engrailed expression and retinotectal projection from a view of plasticity and rigidity of rostrocaudal polarity in the tectum. We produced ectopic tecta by anisochronal transplantations between E3 host and E2 donor, and showed that there is little repressive influence at E3 around the mes-diencephalon junction. We then made chimeric double-rostral tectum (caudal half of it was replaced by rostral half of the donor tectum) or double-caudal tectum at E3. The transplants kept their original staining pattern in hosts. Consequently, the chimeric tecta showed wholly negative or positive staining of engrailed protein on the grafted side. In such tecta retinotectal projection pattern was disturbed as if the transplants retained their original position-specific characters. We propose from these heterotopic and anisochronal experiments that the engrailed expression can be a marker for subsequent rostrocaudal polarity in the tectum, both as regards cytoarchitectonic development and retinotectal projection.

scholarly journals Tau Phosphorylation and Aggregation in the Developing Human Brain

2019 ◽  
Vol 78 (10) ◽  
pp. 930-938 ◽  
Author(s):  
Marco M Hefti ◽  
SoongHo Kim ◽  
Aaron J Bell ◽  
Ryan K Betters ◽  
Kimberly L Fiock ◽  
...  
Keyword(s):  
Staining Pattern ◽  
Molecular Layer ◽  
Fetal Brain ◽  
Tau Phosphorylation ◽  
Developing Brain ◽  
Aging Brain ◽  
Positive Staining ◽  
Western Blots ◽  
Autopsy Brain Tissue ◽  
Tau Aggregation

Abstract Tau hyperphosphorylation, mostly at serine (Ser) or threonine (Thr) residues, plays a key role in the pathogenesis of Alzheimer disease (AD) and other tauopathies. Rodent studies show similar hyperphosphorylation in the developing brain, which may be involved in regulating axonal growth and plasticity, but detailed human studies are lacking. Here, we examine tau phosphorylation by immunohistochemistry and immunoblotting in human fetal and adult autopsy brain tissue. Of the 20 cases with sufficient tissue preservation, 18 (90%) showed positive staining for S214 (pSer214), with the majority also positive for CP13 (pSer202), and PHF-1 (pSer396/pSer404). AT8 (pSer202/pThr205) and RZ3 (pThr231) were largely negative while PG5 (pSer409) was negative in all cases. Immunoblotting showed tau monomers with a similar staining pattern. We also observed phospho-tau aggregates in the fetal molecular layer, staining positively for S214, CP13, and PHF1 and negative for thioflavin S. These corresponded to high-molecular weight (∼150 kD) bands seen on Western blots probed with S214, PHF1, and PG5. We therefore conclude that fetal phosphorylation overlaps with AD in some residues, while others (e.g. T231, S409) appear to be unique to AD, and that tau is capable of forming nontoxic aggregates in the developing brain. These findings suggest that the fetal brain is resilient to formation of toxic aggregates, the mechanism for which may yield insights into the pathogenesis of tau aggregation and toxicity in the aging brain.

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