Immunohistochemistry Cytokeratin 19 Rabbit

Get tips on using Purified anti-Tubulin β-3 (TUBB3) Antibody (Previously Covance catalog# PRB-435P) to perform Immunohistochemistry Mouse - TUBB3

Get tips on using T-PER™ Tissue Protein Extraction Reagent to perform Protein isolation Tissue - Rabbit eye retina/choroids

Get tips on using Anti-Collagen Type VII Antibody, clone 32,-VII to perform Immunohistochemistry Collagen VII [II-32] - Mouse Human -NA-

Get tips on using Monoclonal Mouse Anti-Human Ki-67 Antigen (Dako Omnis) Clone MIB-1 to perform Immunohistochemistry Human - Ki-67

Get tips on using TRIzol™ Plus RNA Purification Kit to perform RNA isolation / purification Cells - primary rabbit aortic endothelial cells

Get tips on using Monoclonal Anti-Glial Fibrillary Acidic Protein (GFAP) to perform Immunohistochemistry Anti-Glial Fibrillary Acidic Protein (GFAP) - Mouse Human -NA-

Get tips on using In Situ Cell Death Detection Kit, TMR red to perform TUNEL assay cell type - Rabbit synovial fibroblasts

Get tips on using anti-alpha-Smooth Muscle Actin mouse monoclonal, ASM-1 to perform Immunohistochemistry Alpha smooth muscle Actin - Mouse -NA- -NA-

Get tips on using Live and Dead Cell Assay (Abcam) to perform Live / Dead assay mammalian cells - rabbit bone marrow mesenchymal stem cells

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining efficacy of transduction and shRNA on its target site.