Assessment of Human Islet Composition and Acinar Cell Component by Immunofluorescence Staining v2

This Standard Operating Procedure (SOP) is based on the Human Islet Phenotyping Program of the IIDP Immunofluorescence Staining Procedure. This SOP provides the HIPP procedure for immunofluorescent staining, imaging, and analysis of islet preparations. This SOP defines the assay method used by the Human Islet Phenotyping Program (HIPP) for quantitative and qualitative determination of the Purified Human Pancreatic Islet product, post-shipment, manufactured for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored research in the Integrated Islet Distribution Program (IIDP).

Silver-Chitosan Nanocomposite Prepared With Aqueous Sodium-hydroxide and Aqueous Acetic Acid Solutions: Characteristics and Their Cytotoxic Effects

In this study cytotoxic effects of silver-chitosan nanocomposites with aqueous sodium-hydroxide solution (SCNC-ASHS), and aqueous acetic acid solution (SCNC-AAAS) were evaluated, in vitro. The morphology of the synthesized nanoparticles were characterized by Fourier-Transform Infrared Spectroscopy (FTIR), and Scanning Electron Microscopy (SEM). Their cytotoxicity were then evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) in concentrations of 1.56 to 400 µg/ml, and acridine orange/ethidium bromide (AO/EB) staining after 24h and 48h. Results showed the cytotoxicity of 400 µg/ml of SCNC-ASHS on Vero and HT-29 cells of 80.57% and 84.37% after 24h, and 82.20% and 84.84% after 48h. While, the values for SCNC-AAAS on Vero and HT-29 cell-lines were respectively 80.63% and 87.64% after 24h, and 83.60% and 87.44% after 48h. The most toxicity on HT-29 cells was belonged to SCNC-AAAS with IC50 of 40.4 µg/ml. In the staining procedure, cell viability for 25 µg/ml concentration of SCNC-AAAS was 41.84% in HT-29 cell and, for 6.25 µg/ml of SCNC-AAAS was 37.51% in Vero cells. A considerable decrease in cell viability was observed. Types of nanoparticles, synthesis methods, and different cell lines play role in inducing cytotoxicity. Anti-cancer effect of the nanoparticles on the colon cancerous cells (HT-29), of that SCNC-AAAS displayed higher effect than SCNC-ASHS.

Variations of Staining Sodium Dodecyl Sulfate–Polyacrylamide Gels with Coomassie Brilliant Blue

Many variations of the original Coomassie Brilliant Blue staining procedure are in use. This protocol describes some selected variations on the standard procedure that give comparable and consistent staining results for proteins in the 20- to 200-kDa range.

MIBI staining v2

This protocol is the standard FFPE tissue staining procedure recommended for Multiplex Ion Beam Imaging Time of Flight instrument (MIBI_TOF) developed in the Sean C. Bendall and Michael R. Angelo labs. The protocol has been successfully used for MIBI and is the result of extensive optimization experiments. It is inspired from state-of-the art of immunohistochemistry staining procedures but differs in some very important steps, namely, glutaraldehyde fixation and final washes prior tissue dehydration. Failure to follow exactly all steps described in this procedure may result in inconsistencies in output data after MIBI_TOF acquisition.

Specific Detection of Endogenous S129 Phosphorylated α-Synuclein in Tissue Using Proximity Ligation Assay

BACKGROUND: Synucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by the accumulation of protein aggregates called Lewy Bodies. Lewy bodies are primarily composed of α-synuclein (asyn) protein, which is phosphorylated at serine 129 (pS129) when aggregated. Currently available commercial antibodies used to stain for pS129 asyn can cross react with other proteins, thus making it difficult to specifically detect endogenous pS129 asyn and to interpret pS129 asyn staining. OBJECTIVE: To develop a staining procedure that detects pS129 asyn with high specificity and low background. METHODS: We use the fluorescent and brightfield in situ Proximity Ligation Assay (PLA) to specifically detect pS129 asyn in cell culture, mouse brain sections, and fixed human brain tissue. RESULTS: The pS129 asyn PLA specifically stained for endogenous, soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. CONCLUSIONS: This PLA method can be used to specifically detect pS129 asyn in order to further explore and understand its role in health and disease.

Prevalence and Pathology of Gastrointestinal Parasites in Free Range Pigs

Background: Gastrointestinal (GI) parasitism in pigs is often associated with subclinical infections leading to poor weight gain and reduced market value. One of the most significant risks is pigs being the host for many zoonotic parasites and thereby threatening human health. Despite the epidemiological data being available from different states of the country, records from Andhra Pradesh are scanty. Hence, a study was conducted to determine the prevalence of gastrointestinal parasites in free range pigs of Proddatur municipality Andhra Pradesh, India. Methods: About 142 fecal samples were collected from free range pigs slaughtered in four localities of Proddatur municipality over a period of six months. The fecal samples were later subjected for parasitological examination and the tissue pieces of intestines collected from the slaughtered animals with the embedded parasites and those with pathological changes were subjected to histological staining procedure for identification. Result: Fecal examination revealed 80.98% (115/142) positivity for parasitic ova or occysts. About eleven species of parasites were identified; of them nine were helminths (83.8%) and two were protozoan (10.5%) parasites. Infection with Ascarops spp. (28.2%) and Fasciolopsis buski (17.6%) was found to be significantly (P less than 0.05) higher. The tissue sections of the intestines with pathological lesions revealed embedded parasites in intestinal mucosa infiltrated with eosinophils and mononuclear cells. The higher prevalence of GI parasites in slaughtered pigs in Proddatur region rises concern towards the impact on the health of pigs and as well as pork consumers suggesting a strategic control for GI parasites in pig farming.

Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin–biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions:

The Contribution of Polysaccharide Antigens From Clinical Proteus spp. and Klebsiella spp. Isolates to the Serological Cross-Reactions

Klebsiella spp. and Proteus spp. cause hospital-acquired urinary tract infections (UTIs), which are often related to the use of catheters. To create a vaccine preventing UTI, immunogenic bacterial antigens with common epitopes are still being looked for. In this work, the role of polysaccharide antigens of four Klebsiella spp. and eight Proteus spp. strains in serological cross-reactions with specific antisera was examined. Enzyme-linked immunosorbent assay (ELISA), Western blotting, and silver staining by Tsai method were performed. The Klebsiella and Proteus spp. LPSs and cells were used as antigens. Polyclonal rabbit sera specific to Klebsiella oxytoca 0.023 and 0.062 strains and four Klebsiella spp. LPSs were obtained. The ELISA and Western blotting results showed the strongest cross-reactions occurring between lipopolysaccharides (LPSs) from four Klebsiella strains and P. vulgaris O42 antiserum. The silver-staining procedure revealed the patterns typical of both slow- and fast-migrating mass species of the Klebsiella LPSs. The Klebsiella spp. antigens also cross-reacted with four P. penneri antisera, and most of the reactions were observed as low-migrating patterns. From two K. oxytoca antisera obtained in this work, only one, the K. oxytoca 0.062 antiserum, cross-reacted with satisfactory strength with P. penneri LPSs (19, 22, and 60). Obtaining cross-reactions between the antigens of Klebsiella strains and Proteus antisera and in the opposite systems is important for proving the immunogenic role of polysaccharide antigens in triggering the immunological response.