scholarly journals Considerations when quantitating protein abundance by immunoblot

2015 ◽  
Vol 308 (6) ◽  
pp. C426-C433 ◽  
Author(s):  
Alicia A. McDonough ◽  
Luciana C. Veiras ◽  
Jacqueline N. Minas ◽  
Donna Lee Ralph
Keyword(s):  
Detection System ◽  
Short Review ◽  
Convincing Evidence ◽  
Protein Abundance ◽  
Tissue Samples ◽  
Signal Density ◽  
Crude Mixture ◽  
Quantitative Analyses ◽  
Protein Staining ◽  
Negative Controls

The development of the immunoblot to detect and characterize a protein with an antisera, even in a crude mixture, was a breakthrough with wide-ranging and unpredictable applications across physiology and medicine. Initially, this technique was viewed as a tool for qualitative, not quantitative, analyses of proteins because of the high number of variables between sample preparation and detection with antibodies. Nonetheless, as the immunoblot method was streamlined and improved, investigators pushed it to quantitate protein abundance in unpurified samples as a function of treatment, genotype, or pathology. This short review, geared at investigators, reviewers, and critical readers, presents a set of issues that are of critical importance for quantitative analysis of protein abundance: 1) Consider whether tissue samples are of equivalent integrity and assess how handling between collection and assay influences the apparent relative abundance. 2) Establish the specificity of the antiserum for the protein of interest by providing clear images, molecular weight markers, positive and negative controls, and vendor details. 3) Provide convincing evidence for linearity of the detection system by assessing signal density as a function of sample loaded. 4) Recognize that loading control proteins are rarely in the same linear range of detection as the protein of interest; consider protein staining of the gel or blot. In summary, with careful attention to sample integrity, antibody specificity, linearity of the detection system, and acceptable loading controls, investigators can implement quantitative immunoblots to convincingly assess protein abundance in their samples.

In situ microprobe quantitation of inorganic particle burden in paraffin sections of lung tissue

1988 ◽  
Vol 46 ◽  
pp. 990-991
Author(s):  
Jerrold L. Abraham
Keyword(s):  
Lung Tissue ◽  
Quantitative Information ◽  
Particulate Material ◽  
Paraffin Sections ◽  
Tissue Samples ◽  
Qualitative And Quantitative ◽  
The Past ◽  
Quantitative Analyses ◽  
Data Result

Inorganic particulate material of diverse types is present in the ambient and occupational environment, and exposure to such materials is a well recognized cause of some lung disease. To investigate the interaction of inhaled inorganic particulates with the lung it is necessary to obtain quantitative information on the particulate burden of lung tissue in a wide variety of situations. The vast majority of diagnostic and experimental tissue samples (biopsies and autopsies) are fixed with formaldehyde solutions, dehydrated with organic solvents and embedded in paraffin wax. Over the past 16 years, I have attempted to obtain maximal analytical use of such tissue with minimal preparative steps. Unique diagnostic and research data result from both qualitative and quantitative analyses of sections. Most of the data has been related to inhaled inorganic particulates in lungs, but the basic methods are applicable to any tissues. The preparations are primarily designed for SEM use, but they are stable for storage and transport to other laboratories and several other instruments (e.g., for SIMS techniques).

scholarly journals Introducing a New Feature to Allergy and Rhinology

2014 ◽  
Vol 5 (1) ◽  
pp. 215265671400500
Keyword(s):  
Case Reports ◽  
Short Review ◽  
Final Diagnosis ◽  
Open Access Journal ◽  
Original Research ◽  
Tissue Samples ◽  
Case Presentation ◽  
New Type ◽  
New Feature ◽  
Case Data

In this issue of Allergy and Rhinology, we are pleased to introduce a new type of article, “Pathology Quiz Case,” to complement the original research articles, case reports, and reviews that we currently publish. This special submission should consist of a case presentation that includes the following elements – 1) patient history, exam and initial case data, 2) pathological description of tissue samples, 3) differential diagnosis, 4) final diagnosis, and 5) a short review of the disease entity and the patient course. This feature should be educational for all trainees and practicing otolaryngologists. In particular, we welcome medical student, resident, and fellow submissions from otolaryngology training programs. Allergy and Rhinology is an open access journal cited in PubMed.

scholarly journals Expression of Maspin and Ezrin Proteins in Periocular Basal Cell Carcinoma

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Mansooreh Bagheri ◽  
Masoomeh Eghtedari ◽  
Mandana Bagheri ◽  
Bita Geramizadeh ◽  
Mohammadreza Talebnejad
Keyword(s):  
Basal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Basal Cell ◽  
Detection System ◽  
Inverse Correlation ◽  
Tissue Samples ◽  
Margin Involvement ◽  
Ezrin Expression ◽  
Maspin Expression ◽  
Periocular Region

Background.The aim of this study was to investigate maspin and ezrin expression in different subtypes of periocular basal cell carcinoma (BCC).Methods.Tissue samples from 43 patients with periocular BCC. Our cases were comprised of 10 morpheaform, 25 nodular, and 8 adenoid type BCCs. Immunohistochemical staining for maspin and ezrin was performed by Envision detection system.Results.There was no difference between different subtypes of BCC in maspin expression regarding positivity, intensity, and pattern of expression. Ezrin was expressed in all subtypes of BCC but the intensity was significantly higher in morpheaform BCC compared to nodular and adenoid types (P<0.001andP=0.012, resp.); ninety percent of morpheaform samples showed strong ezrin intensity, while this strong intensity was only present in 25% and 12% of adenoid and nodular subtypes, respectively. There was no correlation between age, sex, or tumor margin involvement and expression of neither maspin nor ezrin. There was no correlation between maspin and ezrin expression except in nodular type, in which an inverse correlation was found(P=0.004).Conclusion.Ezrin is expressed intensely in morpheaform BCC of periocular region. Further studies are needed to show the significance of this finding in prognosis of morpheaform BCC.

scholarly journals Comparing MTA and Ketac Molar Easymix for Furcation Perforation Repair using a Volumetric Method

2011 ◽  
Vol 1 (1) ◽  
Author(s):  
Sadullah KAYA ◽  
Selengül GANİDAĞLI AYAZ ◽  
Mehmet Sinan DOĞAN ◽  
Haluk AYDIN
Keyword(s):  
Methylene Blue ◽  
Mineral Trioxide Aggregate ◽  
Volume Measurement ◽  
Volumetric Method ◽  
Molecular Characteristics ◽  
Blue Dye ◽  
Dye Penetration ◽  
Quantitative Analyses ◽  
Group 2 ◽  
Negative Controls

Aim: We compared the ability of mineral trioxide aggregate (MTA) and Ketac Molar Easymix (KM) to repair furcal perforations in extracted human molars, based on the volume of methylene blue dye penetration. Methodology: In total, 44 human mandibular molars were divided randomly into two (n = 20 each) experimental groups, with two teeth used as positive controls and two teeth without perforations used as negative controls. Group 1 was repaired with MTA and group 2 with Ketac Molar Easymix. The volumetric determination of dye penetration was based on the molecular characteristics of methylene blue. The standard area of a methylene blue particle is known and the surface area can be calculated. We converted the dye penetration area into a volume and performed quantitative analyses. Results: Volume measurement using the dye penetration method showed that KM resulted in more microleakage than MTA (p < 0.05). Conclusions: Mineral trioxide aggregate resulted in significantly less dye leakage than Ketac Molar Easymix using a volumetric measurement method.   How to cite this article: Kaya S,  Ganidağlı Ayaz S, Doğan MS,  Aydın H. Comparing MTA and Ketac Molar Easymix for furcation perforation repair using a volumetric method. Int Dent Res 2011;1:13-17.  

scholarly journals Increased AQP7 abundance in skeletal muscle from obese men with type 2 diabetes

2018 ◽  
Vol 315 (3) ◽  
pp. E367-E373 ◽  
Author(s):  
Janne Lebeck ◽  
Esben Søndergaard ◽  
Søren Nielsen
Keyword(s):  
Type 2 Diabetes ◽  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Protein Abundance ◽  
Tissue Samples ◽  
Triglyceride Accumulation ◽  
Meal Intake ◽  
Fasting And Refeeding ◽  
Subcutaneous Adipose

Aquaglyceroporin 7 (AQP7) facilitates the transport of glycerol across cell membranes. In mice, fasting and refeeding regulate adipose tissue AQP7 abundance, and a role in controlling triglyceride accumulation in adipose tissue has been proposed. AQP7 is also expressed in skeletal muscle, where its function remains to be determined. Here, the abundance of AQP7 in abdominal subcutaneous adipose tissue (SAT) and skeletal muscle was evaluated in the overnight fasted and postprandial state in eight lean and eight obese men with type 2 diabetes (T2D). A biopsy from SAT and muscle was collected after an overnight fast and 2 h after ingestion of a low-fat test meal. Palmitate turnover was evaluated using a [9,10-3H] palmitate dilution technique. Tissue samples were analyzed by immunoblotting. Meal intake did not affect AQP7 expression in SAT or skeletal muscle. No association between the SAT AQP7 abundance and palmitate turnover was found. SAT AQP7 abundance was similar in lean and obese T2D men, whereas muscle AQP7 abundance was more than fourfold higher in obese T2D men. In conclusion, meal intake did not affect AQP7 protein abundance in SAT or skeletal muscle. In addition, SAT AQP7 expression does not appear to be involved in the regulation of adipose tissue lipolysis. However, in contrast to SAT AQP7, skeletal muscle AQP7 protein abundance is markedly increased in obese T2D men, potentially contributing to the excess lipid accumulation in skeletal muscle in type 2 diabetes.

scholarly journals Enhancement of immunohistochemical detection of Salmonella in tissues of experimentally infected pigs

2015 ◽  
Vol 59 (3) ◽  
Author(s):  
J. Rieger ◽  
P. Janczyk ◽  
H. Hünigen ◽  
J. Plendl
Keyword(s):  
Lymph Nodes ◽  
Detection System ◽  
Intestinal Damage ◽  
Immunohistochemical Detection ◽  
Mesenteric Lymph Nodes ◽  
Tissue Samples ◽  
Serovar Typhimurium ◽  
Triton X ◽  
Foodborne Infections ◽  
Higher Sensitivity

<p><em>Salmonella</em> Typhimurium is one of the main pathogens compromising porcine and human health as well as food safety, because it is a prevailing source of foodborne infections due to contaminated pork. A prominent problem in the management of this bacteriosis is the number of subclinically infected carrier pigs. As very little is known concerning the mechanisms allowing <em>Salmonella</em> to persist in pigs, the objective of this study was to develop an immunohistochemical approach for the detection of salmonellae in tissue of pigs experimentally infected with <em>Salmonella</em> Typhimurium. Samples were obtained from a challenge trial in which piglets of the German Landrace were intragastrically infected with <em>Salmonella</em> enterica serovar Typhimurium DT104 (1.4-2.1x10<sup>10</sup> CFU). Piglets were sacrificed on days 2 and 28 post infection. Tissue samples of jejunum, ileum, colon, ileocecal mesenteric lymph nodes (<em>Lnn. ileocolici</em>), and tonsils (<em>Tonsilla veli palatini</em>) were fixed in Zamboni’s fixative and paraffin-embedded. Different immunohistochemical staining protocols were evaluated. <em>Salmonella</em> was detected in varying amounts in the tissues. Brown iron-containing pigments in the lymph nodes interfered with the identification of <em>Salmonella</em> if DAB was used as a staining reagent. Detergents like Triton X-100 or Saponin enhanced the sensitivity. It seems advisable not to use a detection system with brown staining for bacteria in an experimental setup involving intestinal damage including haemorrhage. The use of detergents appears to result in a higher sensitivity in the immunohistochemical detection of salmonellae.</p>

scholarly journals Protein abundance of urea transporters and aquaporin 2 change differently in nephrotic pair-fed vs. non-pair-fed rats

2012 ◽  
Vol 302 (12) ◽  
pp. F1545-F1553 ◽  
Author(s):  
Raed N. Bou Matar ◽  
Bela Malik ◽  
Xiaonan H. Wang ◽  
Christopher F. Martin ◽  
Douglas C. Eaton ◽  
...  
Keyword(s):  
Urinary Sodium Excretion ◽  
Protein Abundance ◽  
Mrna Levels ◽  
Sprague Dawley ◽  
Tissue Samples ◽  
Aquaporin 2 ◽  
Urine Protein Excretion ◽  
Protein Excretion ◽  
Sprague Dawley Rats ◽  
Ad Libitum

Salt and water retention is a hallmark of nephrotic syndrome (NS). In this study, we test for changes in the abundance of urea transporters, aquaporin 2 (AQP2), Na-K-2Cl cotransporter 2 (NKCC2), and Na-Cl cotransporter (NCC), in non-pair-fed and pair-fed nephrotic animals. Doxorubicin-injected male Sprague-Dawley rats ( n = 10) were followed in metabolism cages. Urinary excretion of protein, sodium, and urea was measured periodically. Kidney inner medulla (IM), outer medulla, and cortex tissue samples were dissected and analyzed for mRNA and protein abundances. At 3 wk, all doxorubicin-treated rats developed features of NS, with a ninefold increase in urine protein excretion (from 144 ± 21 to 1,107 ± 165 mg/day; P < 0.001) and reduced urinary sodium excretion (from 0.17 to 0.12 meq/day; P < 0.001). Urine osmolalities were reduced in the nephrotic animals (1,057 ± 37, treatment vs. 1,754 ± 131, control). Unlike animals fed ad libitum, UT-A1 protein abundance was unchanged in nephrotic pair-fed rats. Glycosylated AQP2 was reduced in the IM base of both nephrotic groups. Abundances of NKCC2 and NCC were consistently reduced (71 ± 7 and 33 ± 13%, respectively) in both nephrotic pair-fed animals and animals fed ad libitum. In pair-fed nephrotic rats, we observed an increase in the cleaved form of membrane-bound γ-epithelial sodium channel (ENaC). However, α- and β-ENaC subunits were unaltered. NKCC2 and AQP2 mRNA levels were similar in treated vs. control rats. We conclude that dietary protein intake affects the response of medullary transport proteins to NS.

The So-called Short-Fiber Controversy: Literature Review and Critical Analysis

2015 ◽  
Vol 139 (8) ◽  
pp. 1052-1057 ◽  
Author(s):  
Victor L. Roggli
Keyword(s):  
Experimental Animal ◽  
Animal Studies ◽  
Scientific Literature ◽  
Scientific Evidence ◽  
Short Fiber ◽  
Convincing Evidence ◽  
Short Fibers ◽  
Human Lung Tissue ◽  
Tissue Samples

Context Numerous articles in the scientific literature indicate that pathogenic fibers with respect to asbestos-related diseases are those that exceed 5 μm in length. Nonetheless, some authors have expressed concerns regarding pathogenicity of shorter fibers. Objective To review the scientific evidence regarding pathogenicity (or lack thereof) of fibers less than or equal to 5 μm in length, with particular attention to publications indicating that such fibers might be hazardous. Data Sources The scientific literature was reviewed for experimental animal studies and human studies that address the role of fiber size in causation of disease. Sources included original studies, as well as review articles related to the topic. Conclusions Experimental animal studies involving inhalation of fibers have demonstrated that fibers greater than 5 μm in length are associated with both pulmonary fibrosis (ie, asbestosis) and malignancies (carcinoma of the lung and mesothelioma). There is no convincing evidence for a pathogenic effect for fibers that are 5 μm or less in length. Fiber analyses of human lung tissue samples provide further support for pathogenicity of long fibers, particularly the more biopersistent amphibole fibers. Similar observations have been reported for nonasbestos mineral fibers. Concerns expressed by some authors (eg, the greater abundance of short fibers) do not alter these conclusions. Similarly, in vitro studies demonstrating biological activity of short fibers do not override inhalational studies of whole animals or the epidemiological findings in humans.

scholarly journals Endothelium Activation during Severe Yellow Fever Triggers an Intense Cytokine-Mediated Inflammatory Response in the Liver Parenchyma

Pathogens ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 101
Author(s):  
Fábio Alves Olímpio ◽  
Luiz Fábio Magno Falcão ◽  
Marcos Luiz Gaia Carvalho ◽  
Jeferson da Costa Lopes ◽  
Caio Cesar Henriques Mendes ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Yellow Fever ◽  
Adhesion Molecule ◽  
Yellow Fever Virus ◽  
Immunohistochemical Analysis ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Hepatic Parenchyma ◽  
Tissue Samples ◽  
Negative Controls

Yellow fever (YF) is a pansystemic disease caused by the yellow fever virus (YFV), the prototype species of the family Flaviviridae and genus Flavivirus, and has a highly complex host-pathogen relationship, in which endothelial dysfunction reflects viral disease tropism. In this study, the in situ endothelial response was evaluated. Liver tissue samples were collected from 21 YFV-positive patients who died due to the disease and five flavivirus-negative controls who died of other causes and whose hepatic parenchyma architecture was preserved. Immunohistochemical analysis of tissues in the hepatic parenchyma of YF cases showed significantly higher expression of E-selectin, P-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and very late antigen-4 in YFV-positive cases than in flavivirus-negative controls. These results indicate that endothelium activation aggravates the inflammatory response by inducing the expression of adhesion molecules that contribute to the rolling, recruitment, migration, and construction of the inflammatory process in the hepatic parenchyma in fatal YF cases.

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