This cytochemistry web page will be updated regularly to describe and illustrate immunocytochemical, affinity cytochemical, and in situ hybridization protocols that are working in the laboratory of Gwen V. Childs, Ph.D. We use these protocols to detect antigens, mRNA and ligands bound to target cells.
Menu of Cytochemical Techniques
 | Affinity cytochemistry detection of ligands bound to target cells. |
| Dual-immunocytochemistry to identify more than one product of a cell at the light microscopic level. |
| In Situ hybridization to detect messenger ribonucleic acids (mRNAs) in a cell. |
| Immunogold detection of two or more antigens at the electron microscopic level. |
| Cool Links to other cytochemistry web pages. |
The use of Affinity cytochemistry to detect ligands bound to target cells.
This protocol is used to detect neuropeptides and growth
factors bound to target sites on pituitary cells. These ligands are first biotinylated in
the laboratory of Brian Miller, Ph.D. (his web page is now under construction). After
tests of their potency and binding affinity, they are used on pituitary cells grown in
culture for 1-5 days (time dependent on the experimental design). The cells are exposed to
physiological concentrations of biotinylated ligands for 1 min to 4 h, depending on the
type of experiment. After exposure, the cells are fixed for light or electron microscopic
detection of the site of binding. The following figure diagrams one of the protocols we
use. 
The protocol described in the following paragraphs detects binding sites for biotinylated Gonadotropin releasing hormone at the light microscopic level. It is diagramed in the above figure. The detection system illustrated in the figure is an Avidin-Biotin-peroxidase complex solution which can be purchased from Vector Laboratories (Elite Kit). It is made 30 min before use. Avidin gold, avidin-ferritin, avidin peroxidase, or avidin fluorescein can also be used, however. If the peroxidase substrate DAB is used, it is nickel intensified and produces a purple-or gray-black reaction product.
The detection protocol is then followed by immunolabeling with a contrasting color reaction product to identify one of the antigens in the target cells. Examples of cells labeled with this dual-labeling protocol are shown below. The first photo shows LH gonadotropes that have bound Biotinylated Growth hormone releasing hormone (GHRH). They express binding sites for GHRH (arrows) during proestrus in the female. Note that the cell in the lower right contains only LH antigens and not Bio-GHRH binding sites. The remaining cells have binding sites for GHRH in patches over the surface. Some of the patches could be in the Golgi complex area. For more information, see the GH web page
Materials:
 |
0.1-10nM Biotinylated D-Lys6 GnRH (dissolve according to Dr. Brian Miller's instructions) |
|
100-1000 nM D-Lys 6 GnRH |
|
13 mm glass coverslips ( Thomas Scientific Catalog number 6672A75) on which pituitary cells are growing (1-5 day cultures) in 24 well trays ( Fisher Scientific , Catalog number 08-757-156). |
|
1:20,000 Anti-LH beta antisera |
|
1:5,000 Anti-FSH beta antisera |
|
ABC Elite kit ( Vector Laboratories , Catalog number PK 6101) |
|
Diaminobenzidine (
Sigma Chemical , Catalog number D 5905) and nickel ammonium sulfate ( Sigma Chemical) for peroxidase detection |
Method:
| transfer coverslips into a new tray with fresh Dulbeccos Modified Eagle's media (DME) ( JRH Biosciences , Catalog number 56499-10L) + 10-4 M ascorbic acid (AA) |
| replace media once with 450 microliters/well DME + AA. |
| add 50 microlitersl/well of Bio-GnRH for 10 mins.(24 degrees or 37 degrees C) (optimal times for maximal labeling will depend on the ligand and how fast it is taken up and/or degraded by the cells) |
| fix cells with 2% glutaraldehyde---30 mins. (room temperature (RT) |
| wash cells with 4.5% sucrose buffer + glycine---4X, 15 mins. each (RT) |
| rinse coverslips with 0.05M phosphate buffer---1X |
| block with phosphate butter containing + 5-10% normal goat serum + 0.1% bovine serum albumin---15 mins. RT |
| incubate with the avidin-biotin peroxidase
complex (ABC) Vector Laboratories , Catalog number PK
6101 ---30 mins. RT |
| wash coverslips with 0.05M phosphate buffer---1X |
| rinse coverslips with 0.05M acetate buffer---2X
|
To detect peroxidase:
| prepare nickel diaminobenzidine---dissolve 0.45 gm. nickel ammonium sulfate in 30 ml. 0.05M acetate buffer |
| add 1 DAB tablet + 20 ul 30% hydrogen peroxide; filtered---6 mins. RT |
| wash coverslips with 0.05M acetate buffer---2X |
Dual-labeling for pituitary antigens
After biotinylated ligands, mRNA or a first antigen is detected we use contrasting colored substrates and immunocytochemistry to detect a second pituitary antigen. The following shows an example of the protocol that detects the second antigen (such as luteinizing hormone or follicle stimulating hormone, or growth hormone). This allows us to identify the hormone content of the cell that binds the ligand, expresses the mRNA or expresses more than one hormone. This figure shows Growth hormone (GH cells) labeled gray or purple-black for biotinylated GnRH and orange for Growth hormone antigens. For more information please see the Growth hormone web page
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 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 |
To detect peroxidase--second reaction (orange-amber)
| prepare orange diaminobenzidine ---dissolve 1 Tris buffer tablet ( Sigma Chemical, Catalog number D 5030.) |
| in 15 ml Millipore filitered water, add 1 diaminobenzidine tablet ( Sigma Chemical, Catalog number D 5905) + 12 ul 30% hydrogen peroxide; filter on Whatman filter paper, use immediately |
| Apply to cells for 5-7 mins. RT |
| wash coverslips with millipore filtered
water---3X dehydrate, dry & permount |
| Warning: diaminobenzidine is eventually washed out in water soluble mounting media, especially temporary mounting media like glycerol. Therefore, if another peroxidase substrate is used, that requires water soluble media, please store the slides dry and use glycerol ONLY! for short periods of time. The best way to store them is dehydrated and mounted in permount. Back to Menu |
Dual-immunocytochemistry at the light microscopic level to detect two antigens in the same cell
This protocol employs rapid cytochemistry kits from DAKO, both of which use peroxidase linked to streptavidin. It is illustrated above. The orange label for LH antigens can be distinguished from the black label for FSH antigens.
The first protocol is as folows:
| cells are grown on coverslips in 24 well trays and fixed in 2.5% glutaraldehyde as in previous protocols. |
| 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
Vendors for Cytochemistry Techniques
| Dako Corporation, 6392 Via Real, Carpenteria, Ca 93013; 800-235-5763. Dimensions has a web page devoted to DAKO Corporation which has access to data sheets |
| Fisher Scientific, 10700 Rockley Road, Houston, Tx 77099; 800-876-1900 or 800-766-7000; 800-395-5442 (instrument service) Link to their Web page @ Fisher Scientific Web page |
| JRH Biosciences, PO Box 14848 Lenexa, KS 66325 800-255-6032 |
| Sigma Chemical, PO Box 14508; St. Louis, MO 63178; 800-325-3010 Link to their Web page at Sigma Chemical Web page . |
| Thomas Scientific, 99 HIgh Hill Road, PO Box 99, Suresdesboro, NJ 08085-0099, 800-345-2100 |
| Vector Laboratories, 30 Ingold Road, Burlingame, Ca 94010, 800-227-6666.
Dimensions has a web page devoted to Vector
Laboratories |
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URL Address: http://www.cytochemistry.net/cytochem.htm
childsgwenv@uams.edu or gvchilds@cytochemistry.net
© copyright 1995 Gwen V. Childs, Ph.D.