scientificprotocols

Authors: Jasdeep Kaur & C. Raman Suri 

Abstract

We report a protocol that employs direct coating of smaller hapten on microtiter ELISA plates for the detection of low molecular weight analytes such as pesticides in an immunoassay format. In this method, the polystyrene surface of microtiter plates was functionalize with amino groups using 3-aminoprpyltriethoxysilane (APTES) for the covalent linkage to small molecular hapten with carboxyl groups. The developed immunoassay format could be used as convenient quantitative tool for the sensitive and rapid screening of pesticides and other low molecular weight analytes in samples.

Introduction

Immunological methods such as enzyme linked immunosorbent assay (ELISA) are increasingly becoming important for pesticides residual analysis due to the high inherent selectivity of detecting molecules, i.e., antibodies. The fact that antibodies could be made virtually against any substance, and its usages in developing highly sensitive assay makes this approac

scientificprotocols

Authors: Joshua Hunsberger & Samuel Newton 

Introduction

Below are the procedures we used for microarray analysis.

Procedure

1. Isolate total hippocampal RNA from individual animals using (RNA Aqueous, Ambion).

• Determine RNA quality by measuring optical density values (260/280). Values should be consistently at or over 1.9.

scientificprotocols

Authors: Joshua Hunsberger & Samuel Newton 

Introduction

Below are detailed procedures for immunohistochemistry and in situ hybridization performed on fresh frozen cryostat cut sections.

Procedure

Immuno on Cyrostat Sections (Cut sections on Cryostat at 14um)

scientificprotocols

Authors: Gang Chen, Ming Jin & Xiao-bing Yuan 

Introduction

Post-mitotic neurons in the developing cortex migrate along radial glial fibers to the proper location in the cortical plate and form the layered structure. Here we report that the radial migration of rat layer II/III cortical neurons requires the guidance of an extracellular diffusible factor Semaphorin-3A (Sema3A). Sema3A is expressed in a descending gradient across the cortical layers, whereas its receptor neuropilin-1 (NP1) is expressed at a high level in migrating neurons. Using in utero eletroporation to down-regulate or conditional knockout of NP1 in newborn cortical neurons impeded their radial migration by disrupting their radial orientation during migration without altering their cell fate. Studies in cultured cortical slices further showed the requirement of the endogenous gradient of Sema3A for the proper migration of newborn neurons. Finally, transwell chemotaxis assays showed that isolated newborn neurons were attracted by

scientificprotocols

Authors: Joshua Hunsberger & Catharine Duman 

Introduction

Below are procedures for administering the forced swim test (mice & rats), tail suspension test, elevate plus maze, open field test, and novelty induced hypophagia.

Procedure

Forced Swim Test (FST) in mice:

scientificprotocols

Authors: Pisana Moroni Rawson, Caroline Molette, Melissa Videtta, Alessandro Sette, Laura Altieri, Debora Franceschini, Tiziana Donato, Marino Paroli, Francesca Meloni, John Sidney & Vincenzo Barnaba 

Introduction

Apoptotic cells, via the activation of caspases, undergo significant proteome alterations. Phagocytosis of apoptotic cells by dendritic cells (DCs) leads to the processing of the apoptotic cell-associated (apoptotic) antigens and the cross-presentation of the resulting peptides on major histocompatibility complex class I molecules. This phenomenon seems crucial for inducing either cross-priming or cross-tolerance of CD8+ T cells, based on the presence or absence of various infectious or danger signals influencing the switch from tolerogenic immature DCs (iDCs) to mature DCs (mDCs) with high stimulatory and migratory capacities.

Here, we demonstrate the following: (a) the protein modifications in apoptotic T cells, as detected by comparing the proteomic profiles of apoptotic and nonapopto

scientificprotocols

Authors: Chien-Sheng Chen & Heng Zhu 

Introduction

We have developed a high-throughput protein purification protocol that allows us to purify ~4,000 proteins within 10 hours from prepared culture. By combining steps of cell lysis and protein capture on affinity resins in sealed filter plates, we reduced the number of pipetting steps and thus, human errors.

Equipment

Q-Fill2

scientificprotocols

Authors: Yoichi Sekita 

Introduction

This is a protocol to observe Rtl1 protein localization in placenta. There are three kinds of trophoblast cells and endothelial cells of fetal capillary in placental labyrinth zone. CD31 is a marker protein of endothelial cells. Therefore, nuclei out of green signal of CD31 are of the trophoblast cells.

Procedure

1. Embed fresh placenta in OCT compound immediately after picking up from uterus.

• Freeze the compound in cooled 2-methyl butane in a liquid nitrogen bath.

scientificprotocols

Authors: Lars Perk, Gerard Visser, Marianne Budde, Maria Vosjan, Paul Jurek, Garry Kiefer & Guus van Dongen 

Introduction

Presently, hundreds of monoclonal antibodies (mAbs) and mAb fragments are under clinical development because of their excellent potential for the systemic treatment of cancer and other pathological conditions (1, 2). Positron emission tomography (PET) offers an exciting imaging option to confirm and quantify selective tumor uptake of such targeting molecules (3).

To enable PET imaging of mAbs (immuno-PET), an appropriate positron emitter, with a half-life (t1/2) that is compatible with the time needed to achieve optimal tumor-to-nontumor ratios (typically 2-4 days for intact mAbs), has to be securely coupled to the targeting molecule. For this purpose we recently described the large scale production of pure zirconium-89 (89Zr; t1/2: 78.4 h) and a strategy for labeling mAbs with 89Zr via a multi-step synthesis using a succinylated-derivative of desferrioxamine B (Df) as bifuncti

scientificprotocols

Authors: Mariarosaria Musar 

Introduction

We describe a very simple method to immunostain Drosophila mitotic chromosomes for spindle checkpoint proteins and kinetochore components. By using appropriate primary antibodies, this procedure allowed us to detect Zw10, Zwilch, Cenp-C, Cenp-meta and BubR1 on kinetochores of larval brain chromosomes. However, the same method can be used for detecting also structural components of chromosomes (our unpublished results).

Reagents

1. Saline (0.7% NaCl),

• Hypotonic solution (0.5% sodium citrate).