Vascularization of primary and secondary ossification centres in the human growth plate.

Background: The switch from cartilage template to bone during endochondral ossification of the growth plate requires a dynamic and close interaction between cartilage and the developing vasculature. Vascular invasion of the primarily avascular hypertrophic chondrocyte zone brings chondroclasts, osteoblast- and endothelial precursor cells into future centres of ossification.Vascularization of human growth plates of polydactylic digits was studied by immunohistochemistry, confocal-laser-scanning-microscopy and RT-qPCR using markers specific for endothelial cells CD34 and CD31, smooth muscle cells α-SMA, endothelial progenitor cells CD133, CXCR4, VEGFR-2 and mesenchymal progenitor cells CD90 and CD105.

Nonradioactive detection of differentially expressed genes using complex RNA or DNA hybridization probes.

The analysis of differential gene expression has become increasingly important in recent years. Typically, differentially expressed genes are identified in a primary screening procedure, yielding candidate genes whose differential expression has to be verified. We provide a highly sensitive, efficient and nonradioactive differential screening procedure to analyze numerous candidate genes in a single step.

Nonradioactive Northern blot analysis of plant RNA and the application of different haptens for reprobing.

Northern blot analysis using radioactive probes is still the most common technique to determine the accumulation of transcripts in cells and tissues. The main disadvantages of this technique are the possible health hazard, inconvenience during handling, the high amount of RNA target necessary for detection, and difficulties with stripping and reprobing. In this paper, we propose an easily applicable protocol for Northern blot analysis of plant RNA with enhanced sensitivity using digoxigenin-, fluorescein-, or biotin-labeled in vitro transcripts derived from PCR products.

Experimental procedure for the detection of a rare human mRNA with the DIG System.

Newcomers to the DIG System often inquire about the possibility of performing Northern blot hybridizations with nonradioactive techniques. With the following examples, we would like to share our protocol for performing highly sensitive Northern blots. This procedure strictly adheres to the standard procedures detailed in our manuals and pack inserts, and there are no special "tricks" required.

Detection of single-copy sequences with digoxigenin-labeled probes in a complex plant genome after separation on pulsed-field gels.

In recent years, the application of rare cutting restriction enzymes, the separation of resulting DNA fragments on pulsed-field gels and the subsequent Southern blot analysis using radioactively labeled probes have been standard laboratory methods to create long-range physical maps of complex genomes. The disadvantages of this technology are the hazardous handling risks when working with radioactivity and long exposure times. In this paper, we describe the use of nonradioactively labeled probes for single-copy sequence detection in a complex plant genome after pulsed-field electrophoretic separation of DNA fragments in the Mbp range.