Lab Protocols
In Vitro Production Protocol
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In Vitro Production of Bovine Embryos
The procedures for in vitro production [IVP; i.e. in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of embryos described here are based on procedures developed in other laboratories at the University of Wisconsin (Neal First), and University of Missouri (R. Michael Roberts) and University of Guelph (W. Allan King).
Keep in mind that the protocols described here are not fixed but rather constantly evolve as new developments take place. Therefore, practitioners of IVP will be well advised to experiment with the procedures used, especially after reading of improvements made by other laboratories.
The protocol is organized as a series of pdf files that contain information for setting up the laboratory, procedures for conducting IVP, and other useful protocols for IVP. The combined manual was produced by merging all the individual files. Access the IVP Manual Here.
Other Useful Information
- Bovine Embryo Book
- Video: Oocyte Slashing
- Video: Oocyte Selection for Bovine IVF (searching)
- Video: Making Drops
- Video: Picking up Embryos
In addition, links to other information that will be helpful for doing IVP (images, videos, other embryo protocols, etc.) are located below
- IVF Docs
Immunochemical Procedures
Embryos
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Apoptosis
- FAM Caspase 8 and 9 Binding Assay for Embryos
- Group II Caspase Activity in Bovine Embryos
- TUNEL Procedure for Preimplantation Embryos
- see Tutorial ImageJ for Luciano Bonilla's guide for using ImageJ for counting cell number.
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Cell Counting
- Cell Number Counting in Preimplantation Embryos
- Live/Dead Stain for Embryos and Oocytes
- Differential staining of trophectoderm and inner cell mass of bovine embryos
- see Tutorial ImageJ for Luciano Bonilla's guide for using ImageJ for counting cell number.
- Immunolocalization of CDX2
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Other
- Detection of reactive oxygen species in preimplantation bovine embryos
- Determining lipid content in embryos using Nile Red fluorescence
- Immunodetection of 5-methylcytosine in preimplantation embryos
- Immunolabeling of embryos for GATA6 and CDX2
- Immunolabeling of embryos for GATA6, NANOG and CDX2
- Isolation of trophectoderm and inner cell mass from bovine blastocysts using magnetic activated cell sorting
- Mini-Chamber for regulating gaseous environment during culture
- Preparation of in-vitro-produced bovine embryos for transfer into recipients
Immunochemical Procedures
Molecular Biology
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Molecular Biology
- Direct cDNA synthesis and pre-amplification of single embryos for RT-PCR
- Sexing bovine preimplantation embryos by PCR - Yao Xiao, Froylan Sosa and Peter J Hansen
- Polymerase Chain Reaction - a detailed guide by Justin Fear
- PCR using the Bio Rad CFX96 - a practical guide by Kyle Dobbs
- RNA purification using Trizol
- Submitting amplicons for sequencing
- PCR-based assay for genotyping of the slick mutation in cattle
Tissue and Cell Culture
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Tissue and Cell Culture
- Culture of endometrial explants
- Culture of BEND cells (a bovine endometrial cell line)
- Establishment of bovine fibroblasts from fetal skin
- Hemacytometer (the use of)
Other Procedures
Other Links
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Other Useful Information
- All videos from the Hansen laboratory can be found at the Peter Hansen YouTube Channel at https://studio.youtube.com/channel/UCuR_cAdHEc6pEAo9f3cX1hg
- Buffer Design-For those who have trouble designing buffers and calculating molarity, here is the site for you (from Professor Rob Benyon at the University of Manchester
- Immunoportal - everything immunohistochemical
- Open Wetware - From MIT, the site "is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering".
- Power Calculations - from R.V. Lenth, University of Iowa
- Protocols Online An excellent source of protocols for biological research
- UNC Tissue Culture Facility - a great site that includes some tips on recognizing and dealing with contamination
- WWW Virtual Library of Cell Biology - Methods, Protocols, and Software A good collection of links to lab protocols