The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Flow Cyt: Use at an assay dependent dilution.
IHC-P: Use at a concentration of 2 - 4 µg/ml for 30 min at RT. No special pretreatment is required for the immunohistochemical staining of formalin/paraffin tissues.
IHC-Fr: Use at a concentration of 2 - 4 µg/ml for 30 min at RT.
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
Binds peptides derived from antigens that access the endocytic route of antigen presenting cells (APC) and presents them on the cell surface for recognition by the CD4 T-cells. The peptide binding cleft accommodates peptides of 10-30 residues. The peptides presented by MHC class II molecules are generated mostly by degradation of proteins that access the endocytic route, where they are processed by lysosomal proteases and other hydrolases. Exogenous antigens that have been endocytosed by the APC are thus readily available for presentation via MHC II molecules, and for this reason this antigen presentation pathway is usually referred to as exogenous. As membrane proteins on their way to degradation in lysosomes as part of their normal turn-over are also contained in the endosomal/lysosomal compartments, exogenous antigens must compete with those derived from endogenous components. Autophagy is also a source of endogenous peptides, autophagosomes constitutively fuse with MHC class II loading compartments. In addition to APCs, other cells of the gastrointestinal tract, such as epithelial cells, express MHC class II molecules and CD74 and act as APCs, which is an unusual trait of the GI tract. To produce a MHC class II molecule that presents an antigen, three MHC class II molecules (heterodimers of an alpha and a beta chain) associate with a CD74 trimer in the ER to form an heterononamer. Soon after the entry of this complex into the endosomal/lysosomal system where antigen processing occurs, CD74 undergoes a sequential degradation by various proteases, including CTSS and CTSL, leaving a small fragment termed CLIP (class-II-associated invariant chain peptide). The removal of CLIP is facilitated by HLA-DM via direct binding to the alpha-beta-CLIP complex so that CLIP is released. HLA-DM stabilizes MHC class II molecules until primary high affinity antigenic peptides are bound. The MHC II molecule bound to a peptide is then transported to the cell membrane surface. In B-cells, the interaction between HLA-DM and MHC class II molecules is regulated by HLA-DO. Primary dendritic cells (DCs) also to express HLA-DO. Lysosomal miroenvironment has been implicated in the regulation of antigen loading into MHC II molecules, increased acidification produces increased proteolysis and efficient peptide loading.
Belongs to the MHC class II family. Contains 1 Ig-like C1-type (immunoglobulin-like) domain.
Ubiquitinated by MARCH1 or MARCH8 at Lys-244 leading to down-regulation of MHC class II. When associated with ubiquitination of the beta subunit of HLA-DR: HLA-DRB4 'Lys-254', the down-regulation of MHC class II may be highly effective.
Cell membrane. Endoplasmic reticulum membrane. Golgi apparatus > trans-Golgi network membrane. Endosome membrane. Lysosome membrane. Late endosome membrane. The MHC class II complex transits through a number of intracellular compartments in the endocytic pathway until it reaches the cell membrane for antigen presentation.
IHC image of HLA DR staining in Human Hodgekins lymphoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab80658, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.