Rabbit Anti-Human CNN1 Polyclonal Antibody

Background Haematoxylin and eosin (H&E)-which respectively stain nuclei blue and other cellular and stromal material pink-are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing and H&E staining of tissues provides challenges with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique to analyse H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. Methods Herein, we utilize heat extraction, physical disruption, and in column digestion for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm(3)) and human lung blood vessels (0.094 mm(3)) from FFPE-fixed H&E-stained sections from Idiopathic Pulmonary Fibrosis (IPF) specimens (n = 3 IPF specimens) were then subject to a qualitative and then quantitative proteomics approach using BayesENproteomics. In addition, we tested the sensitivity of this method by processing and analysing a range of micro-dissected human lung blood vessel tissue volumes. Results This approach yields 1252 uniquely expressed proteins (at a protein identification threshold of 3 unique peptides) with 892 differentially expressed proteins between these regions. In accord with prior knowledge, our methodology approach confirms that human lung blood vessels are enriched with smoothelin, CNN1, ITGA7, MYH11, TAGLN, and PTGIS; whereas morphologically normal human lung alveoli are enriched with cytokeratin-7, -8, -18, -19, 14, and -17. In addition, we identify a total of 137 extracellular matrix (ECM) proteins and immunohistologically validate that laminin subunit beta-1 localizes to morphologically normal human lung alveoli and tenascin localizes to human lung blood vessels. Lastly, we show that this micro-proteomics technique can be applied to tissue volumes as low as 0.0125 mm(3). Conclusion Herein we show that our multistep sample preparation methodology of LCM-MS can identify distinct, characteristic proteomic compositions of anatomical features within complex fixed and stained tissues.

The increased use of cannabis as a therapeutic drug in recent years has raised some concerns due to its potential effects on reproductive health. With regards to the male, the endocannabinoid system is involved in the spermatogenesis and in the sperm function. The chronic use of tetrahidrocannabinol (THC) has been associated with sperm anomalies, decreased sperm motility and structural changes in the testis. However, whether THC affects sperms ability to fertilize and to generate embryos remains unclear. The aim of this study was to evaluate this effect using a mice model of THC chronic treatment. For this purpose, a chronic treatment with THC was carried out. Mice were randomly allocated into two groups: an experimental group treated with a daily dose of 10 mg/kg-body weight THC for a period of 30 days and a control group treated with a vehicle. The THC-mice cortex showed a significant decrease of mRNA of Cnr1 compared to control-mice while, in the testis, the expression of Cnr1 was not affected. The weight of testis and epididymis and the histological analysis did not show any change between groups. On the other hand, no changes were observed in the sperm motility or the sperm concentration. The chronic use of THC did not generate any methylation change in the three CpG regions of Cnn1 analysed, neither in the brain nor in the embryos generated by in vitro fertilization (IVF). Finally, the embryo production by IVF was no different using spermatozoa from both THC and control mice. This work contradicts the belief that THC consumption has a negative effect on male reproductive processes.