Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods.

Abstract

We developed an immunohistochemical method for visualization of vitamin D (VDR) and estrogen receptors (ER) in cryostat sections, using monoclonal antibodies (MAb) to the vitamin D receptor and estrogen receptor, respectively. This method is based on an avidin-biotin labeling technique (LAB). To establish a reliable and sensitive method which can be used easily as a routine diagnostic procedure, we systematically compared four different immunoenzymatic methods with respect to their efficiency in detecting vitamin D and estrogen receptors. Compared to the indirect bridged avidin-biotin (IBRAB), the peroxidase- anti-peroxidase (PAP), and the avidin-biotin complex (ABC) methods, the LAB method produced stronger staining intensities and had higher detection efficiency for both vitamin D and estrogen receptors. In addition, the LAB method had a higher spatial resolution compared to the ABC technique in detection of VDR in normal human skin biopsies. In the case of steroid receptors, i.e., nuclear antigens, immunohistochemistry must deal with a relatively low number of antigenic sites per cell, restricted accessibility of the antigens, and slight differences in antigen concentrations among cells. Under these particular conditions, the chemical properties of the conjugates used in the LAB method may explain why it is superior to the other methods. Consequently, the LAB method is recommended for visualization of ER and VDR.

Identification of Amino Acid Sequence in the Hinge Region of Human Vitamin D Receptor That Transfers a Cytosolic Protein to the Nucleus

“…To modulate the transcription of target genes in response to its cognate ligand 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), VDR must be localized in nucleus and then bind to an enhancer designated as the vitamin D-responsive element (VDRE), forming a heterodimer with retinoid X receptor (1)(2)(3)(4)(5)(6). In contrast to the case for the glucocorticoid receptor (GR), which translocates from the cytoplasm to the nucleus when exposed to its ligand, VDR does not bind to heat shock protein 90, and both immunocytochemical and biochemical fractionation studies suggested the nuclear localization of VDR even in the absence of 1,25(OH) 2 D 3 (7)(8)(9)(10).…”

Influence of different calcium supplies and a single vitamin D injection on vitamin D receptor and calbindin D9k immunoreactivities in the gastrointestinal tract of goat kids

“…Negative controls were performed using tris-buffered saline (TBS) instead of primary antibody and positive controls employing duodenal cross-sections of pigs (cross-reactivity proven and giving identical results; Milde et al, 1989;Schröder et al, 2001). In every tissue specimen, .…”

Immunohistochemical Demonstration of Vitamin D Receptor Distribution in Goat Intestines

“…Following the avidin-biotin block (20 min each, Vector Laboratories, Burlingame, Calif., USA), a serum-free protein block (10 min, DakoCytomation, Zug, Switzerland) was kept in a humidified chamber. Sections were rinsed in TBS and incubated for 80 min with a biotinylated rat monoclonal antibody (9A7 ␥ , NeoMarkers) directed against chicken VDR protein (aa 89-105) demonstrating cross-reactivities with pig and sheep VDR [Milde et al, 1989;Schröder et al, 2001]. This step was followed by rinsing in TBS (5 min), incubation with StreptABComplex/HRP (30 min, DakoCytomation), rinsing in TBS (5 min) and incubation with diaminobenzidine tetrahydrochloride chromogen and H 2 O 2 (Dakocytomation) for 3 min and rinsing with TBS (5 min).…”

“…There is growing direct evidence that the traditional nuclear VDR may also have a unique, non-transcriptional role in plasma membrane-initiated signalling [Fleet, 2004]. This would to some extent explain the detection of VDR in the cytoplasm of target cells [Feldman et al, 1979;Milde et al, 1989;Barsony et al, 1997;Zineb et al, 1998;Prüfer et al, 2000;Horst et al, 2003] -a locus where the receptor protein is synthesized.…”