Dual Labeling for pituitary hormones or receptors.
Once target cells have been detected by affinity cytochemistry with biotinylated analogs, or mRNA has been detected by in situ hybridization, one can dual-label for the protein with immunoperoxidase and a contrasting color substrate. This is done after the peroxidase step is completed in the first protocol (with nickel-intensified DAB). Ideally this should be done immediately after the detection of the biotinylated analog or the mRNA.
Method
- Rinse coverslips with 0.05M Tris buffered saline---1X
- Block again with 0.05M Tris buffered saline + 1% Bovine serum albumin---15 mins. room temperature
- Incubate with either Primary antiserum (i.e. 1:20K Anti-LH/1:5K Anti-FSH)---30 mins. at 37 degrees
- Wash coverslips with 0.05M Tris buffered saline---3X
- Incubate with Biotinylated Goat anti-rabbit IgG ( Vector Laboratories, Catalog number BA-1000) ( 25 microliters stock Biotinylated-IgG + 25 microliters Normal goat serum in 2 milliliters of buffer )---20 mins., room temperature
- Wash coverslips with 0.05M Tris buffered saline---2X
- Incubate with 1:200 peroxidase conjugated streptavidin ( DAKO Corp, Catalog P-397)---20 mins. room temperature
- Wash coverslips with 0.05M Tris buffered saline—2X
- Detect peroxidase with Orange-amber DAB, using the Diaminobenzidine kit, according to kit instructions.
Dual-Immunoperoxidase cytochemistry: Cells in culture
See examples in the Side-bar. Cells are grown on coverslips in 24 well trays and fixed in 2.5% glutaraldehyde as in previous protocols. This protocol detects two antigens in a field. The nickel-intensified DAB detects immunolabeling in the first protocol and the amber DAB detects immunolabeling in the second protocol. This figure shows black-brown labeling for leptin receptor and orange/amber labeling for luteinizing hormone.
Method
- Rinse coverslips with 0.05M Tris buffered saline---1X
- Block with 0.05M Tris buffered saline + 1% Bovine serum albumin( Sigma Chemical, Catalog number A-7638)---15 mins. room temperature
- Incubate with either 1:5,000-100,000 Antiserum to the first antigen---30 mins. at 37 degrees.
- Wash coverslips with 0.05M Tris buffered saline---3X
- Incubate with Biotinylated Goat anti-rabbit IgG ( Vector Laboratories, Catalog number BA-1000) ( 25 microliters stock Biotinylated-IgG + 25 microliters Normal goat serum in 2 milliliters of buffer)---20 mins., room temperature
- Wash coverslips with 0.05M Tris buffered saline---2X
- Incubate with 1:200 peroxidase conjugated streptavidin ( DAKO Corp, Catalog number P-397)---20 mins. room temperature
- Wash coverslips with 0.05M Tris buffered saline---2X
- For the second antigen, the cells are exposed to the same sequence as described in the Dual-labeling protocol under affinity cytochemistry.
For more information, contact:
Gwen Childs, Ph.D.,FAAA
Professor and Chair
Department of Neurobiology and Developmental Sciences
University of Arkansas for Medical Sciences
Little Rock, AR 72205
For questions, contact this email address:
