hansen Protocols

Category: Protein Protocols: Immunoprecipitation Protocols

Antibody-antigen complexes are removed from solution by addition of an insoluble form of an antibody binding protein such as Protein A, Protein G or second antibody. Immunoprecipitation protocols / methodology and technical background information. P.J. Ha - [Read Analysis of Proteins by Immunoprecipitation]

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

Protocol used to for immunohistochemistry on paraffin-embedded sections. Based on use of microwave energy to effect antigen retrieval. The immunohistochemistry procedure, is for use of Biomeda's HistoScan kit based on a streptavidin-peroxidase/biotinylated second antibody detection system with 3-amino, 9-ethylcarbazole (AEC) as chromogen. Undoubtedly, other kits or home-made reagents will also work . - [Read Antigen Retrieval for Immunohistochemistry with Paraffin-Embedded Tissues Protocol]

Category: Cell Culture Protocols

Protocol for culture of BEND cells (Bovine Endometrial Cells). Includes: Culture medium, Thawing Cells, Trypsinazation/ Splitting and freezing. - [Read Culture of BEND Cells (Bovine Endometrial Cells) Protocol]

Category: Cell Culture Protocols

Protocol used to study secretion of proteins and prostaglandins by endometrium from the cow, ewe, mare, bitch and other species. The technique is also useful for culture of peri-implantation conceptuses and placental tissues for metabolic labelling studies and to obtain conceptus secretory proteins for biological studies.The medium used is called Pig MEM, which is a modified minimum essential medium supplemented with non-essential amino acids, vitamins, insulin and additional glucose. - [Read Culture of Endometrial Explants and Peri-implantation Conceptuses to Monitor Synthesis and Secretion]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols

Protocol for extraction and purification of total RNA using TRIzol OR TRI reagent. Includes: Homogenization for Cell Suspensions; Phase Separation; RNA Precipitation; RNA Wash;  Redissolving the RNA; Determination of RNA Concentration and Purity; Preparation of Rnase-free water. - [Read Extraction and Purification of Total RNA using TRIzol OR TRI Reagent Protocol]

Category: Developmental Biology: Embryology Protocols

The FAM caspase binding assay kits from ATCC Corporation can be used to determine amounts of active caspases in cells. The FAM-labeled caspase inhibitor can freely diffuse into the cell. Active caspase irreversibly binds the inhibitor. Upon washing the cells, the amount of fluorescence is proportional to the amount of active caspase in the cell. FAM-LETD-fmk (catalog no. 30-1306) is used to detect caspase 8 and FAM-LEHD-fmk (catalog no. 30-1308) is used for caspase 9. - [Read Fam Caspase 8 and 9 Binding Assay for Embryos Protocol]

Category: Protein Protocols: Lowry Protein Assay

Lowry Protein Assay. The Lowry procedure is one of the most venerable and widely-used protein assays, being first described in 1951 [Lowry et al., J. Biol. Chem. 193: 265-275 (1951)]. Under alkaline conditions, copper complexes with protein. When folin phenol reagent (phospho-molybdic-phosphotungstic reagent) is added, the Folin-phenol reagent binds to the protein. Bound reagent is slowly reduced and changes color from yellow to blue. P.J. Hansen, Dept. of Animal Sciences, University of Florida. - [Read Lowry Protein Assay]

Category: Developmental Biology: Embryology Protocols

Protocol for micro-glutathione assay. - [Read Micro-Glutathione Assay Protocol]

Category: Developmental Biology: Embryology Protocols

A simple and inexpensive chamber for regulating gaseous environment of small culture plates, such as those used for culture of preimplantation embryos, can be constructed using disposable media-filtration devises such as Corning’s 115-ml system. The following is a description of how to make such a device. - [Read Mini-Chamber for Regulating Gaseous Environment During Culture]

Category: Developmental Biology: Embryology Protocols

Procedures for in vitro production of bovine embryos. Includes: Collection of Ovaries; Oocyte Collection; Preparation of IVF Medium; In Vitro Fertilization; Culture of Embryos. - [Read Procedures for In Vitro Production of Bovine Embryos]

Category: Developmental Biology: Embryology Protocols

Protocol to Count Cell Number of Preimplantation Embryos using Nuclear Staining with Hoechst 33342 or DAPI. Includes: Preparation of Embryos; Preparation of Hoechst 33342 dye; Preparation of DAPI; Staining the Embryo; Mounting Embryos to Slides; What to Do When There are Too Many Cells to Count. - [Read Protocol to Count Cell Number of Preimplantation Embryos]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: TUNEL assay apoptosis

TUNEL procedure protocol in Bovine Embryos. Paula-Lopes et al. - [Read TUNEL in Bovine Embryos]

Category: Protein Protocols: Protein Quantitation Concentration Protocols: Bradford Protein Assay Protocols

Use of the Bradford Protein Assay in a Microtiter Plate Format Protocol. Includes a short introduction to the Bradford assy, materials and procedure. - [Read Use of the Bradford Protein Assay in a Microtiter Plate Format]

Category: Protein Protocols: Western Blot Protocol

Western Blotting Using Chemiluminescence. P.J. Hansen. - [Read Western Blotting Using Chemiluminescence]