Other Lab Protocols

List of Protocols

Protocols that are added or revised as of August 2019 are being uploaded into protocols.io. This site is a collection of searchable and citeable protocols.

Assays

Glutathione-a microssay for determining glutathione content in cells (.pdf 247KB)
Bradford Protein Assay (.pdf 152KB)
Lowry Protein Assay (.pdf 17367KB)

Embryos

In Vitro Production of Bovine Embryos

Apoptosis

FAM Caspase 8 and 9 Binding Assay for Embryos (.pdf 48KB)
Group II Caspase Activity in Bovine Embryos (.pdf 133KB)
TUNEL Procedure for Preimplantation Embryos (.pdf 263KB)
- see Tutorial ImageJ (.pdf 249KB) for Luciano Bonilla's guide for using ImageJ for counting cell number.

Cell Counting

Cell Number Counting in Preimplantation Embryos (.pdf 179KB)
Live/Dead Stain for Embryos and Oocytes (.pdf 163KB)
Differential staining of trophectoderm and inner cell mass of bovine embryos (.pdf 270KB)
- see Tutorial ImageJ (.pdf 249KB) for Luciano Bonilla's guide for using ImageJ for counting cell number.
Immunolocalization of CDX2 (.pdf 99KB)

Other

Detection of reactive oxygen species in preimplantation bovine embryos (.pdf 41KB)
Determining lipid content in embryos using Nile Red fluorescence (.pdf 36KB)
Immunodetection of 5-methylcytosine in preimplantation embryos (.pdf 219KB)
Immunolabeling of embryos for GATA6 and CDX2 (.pdf 65KB)
Immunolabeling of embryos for GATA6, NANOG and CDX2 (.pdf 86KB)
Isolation of trophectoderm and inner cell mass from bovine blastocysts using magnetic activated cell sorting
Mini-Chamber for regulating gaseous environment during culture (.pdf 223KB)
Preparation of in-vitro produced bovine embryos for transfer into recipients (.pdf 198KB)

Protocol for collection of embryos for molecular biology experiments

Immunochemical Procedures

Antigen retrieval for immunohistochemistry with paraffin-embedded tissues (.pdf 298KB)
Immunoprecipitation (.pdf 153KB)
Semi-dry transfer of proteins for Western blotting (.pdf 96KB)
Western blotting using ECL reagent (.pdf 99KB)

Molecular Biology

Direct cDNA synthesis and pre-amplification of single embryos for RT-PCR dx.doi.org/10.17504/protocols.io.6knhcve

Sexing bovine preimplantation embryos by PCR - Yao Xiao, Froylan Sosa and Peter J Hansen)

Polymerase Chain Reaction - a detailed guide by Justin Fear (.pdf 818KB)

PCR using the Bio Rad CFX96 - a practical guide by Kyle Dobbs (.pdf 796KB)
RNA purification using Trizol (.pdf 143KB)
Submitting amplicons for sequencing (.pdf 758KB)
PCR-based assay for genotyping of the slick mutation in cattle

Tissue and Cell Culture

Culture of endometrial explants
Culture of BEND cells (a bovine endometrial cell line) (.pdf 100KB)
Establishment of bovine fibroblasts from fetal skin (.pdf 89KB)
Hemacytometer, (the use of) (.pdf 174KB)

Other Procedures

Stain for sperm acrosomes
pH meter (Accumet XL15): Protocol for use in PJH lab (.pdf 153KB)
- Manual
Rectal palpation of cattle (.pdf 89KB)
Synthesis of glutathione ester (.pdf 98KB)

Other Links

Apoptosis Protocols-part of the Immunohistochemistry World Website
Buffer Design-For those who have trouble designing buffers and calculating molarity, here is the site for you (from Professor Rob Benyon at University of Manchester
Immunohistochemistry Protocols from Immunohistochemistry World
Immunoportal - everything immunohistochemical
Molecular Biology Protocols
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, includes some tips on recognizing and dealing with contamination
University of Pennsylvania School of Dental Medicine Flow Cytometry Facility
WWW Virtual Library of Cell Biology - Methods, Protocols, and Software A good collection of links to lab protocols

dobbs

Blastocyst labeled with anti-5-methylcytosine (green) and propidium iodine (courtesy-Kyle Dobbs

ivf_cow

Cow born using IVF protocol