publication . Thesis

Evaluation of cellular processes and identification of candidate genes critical to corneal epithelial development

Nowak-Musial, Magdalena Maria;
Open Access English
  • Country: United Kingdom
Abstract
The overall aim of this study was to determine factors and mechanisms that underlie the regulation of epithelial patterning and homeostasis during corneal development. Histological staining was performed in chick corneas, embryonic day (ED) 4 to 21, to evaluate changes in the overall epithelial cell morphology, in particular cell shape, cell size and the number of epithelial cell layers. Epithelial differentiation patterns were identified in frozen sections of chicken corneas after immunolocalisation of pan-cytokeratins (Pan-CK) and cytokeratin 3 (CK3). Proliferating Cell Nuclear Antigen (PCNA) and caspase 3 (active) immunolocalisation studies, as well as, TUNEL...
Subjects
Medical Subject Headings: sense organs
free text keywords: RE
Related Organizations
118 references, page 1 of 8

2.6.2 Immunolocalisation of caspase 3 (active)

2.7 Image capture

2.8 Statistical analysis

2.9 Western Blotting 2.9.1 Sample preparation 2.9.2 Protein extraction 2.9.3 Protein quantification 2.9.4 Preparation of protein samples 2.9.5 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 2.9.6 Transfer of proteins onto the nitrocellulose membrane 2.9.7 Blocking non-specific binding 2.9.8 Incubation with the primary antibody 2.9.9 Incubation with the secondary antibody 2.9.10 Controls 2.9.11 Visualisation of the specific protein antigens usingECL+™ 2.9.12 Stripping the membranes 2.9.13 Analysis of Western Blotting results

2.10 Preparation of samples for microarray analysis 2.10.1 Isolation of corneal epithelium andRNA stabilisation 2.10.2 RNA extraction from tissue 2.10.3 Optimisation of the protocol for RNAextraction 2.10.4 RNA quantitative and qualitative analysis

2.11 Microarrays preparation 2.11.1 Preparation of Spike-in Controls and T7-Oligo (dT) Primer/Poly-A Controls Mix 2.11.2 First round RNA amplification 2.11.3 IVT labelling reaction 2.11.4 Cleanup of biotin-labelled cRNA 2.11.5 Fragmentation of cRNA 2.11.6 GeneChip® hybridisation 2.11.7 Probe array wash and stain 2.11.8 Scanning of GeneChip®

2.12 Standard and semiquantitative Real-TimePCR 2.12.1 cDNA synthesis 2.12.2 Primers 2.12.3 Standard PCR 2.14.4 Quantitative PCR 2.14.5 DNA agarose gel electrophoresis 2.12.6 Analysis of RT-qPCR

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