Cytochemical Detection of mRNA by in situ hybridization
The following page describes the current favorite
non-radioactive in situ hybridization technique used by Dr.
Gwen V. Childs at the University of Arkansas
for Medical Sciences to
detect mRNAs in whole pituitary cells grown in culture. We no longer use the avidin-biotin
peroxidase complex to detect the biotinylated probes. Instead, we use a new five-step
immunolabeling protocol that detects biotin conjugated to the mRNA probes with
anti-biotin. The protocol can be followed by a second detection system for an
antigen. We use biotinylated antisense oligonucleotide probes
exclusively from GeneDetect.com Contact them at
www.GeneDetect.com.
We are constructing a page that describes the dual-in
situ protocol for biotinylated and digoxygenin labeled probes.
What's new at this site: March 2006
- reference to our use of www.GeneDetect.com for probes
- new diluent for proteinase-K. Important improvement in sensitivity with only 5-10 micrograms/ml. Buffer should be relatively fresh.
- Updated CV with new references
at this site and at
Childs_CV.htm
The figure shows cells labeled with dark blue-black for Growth hormone mRNA (arrows). The labeling for the mRNA is in patches or lines in the cells. At the electron microscopic level (see below), the label is associated with dilated profiles of rough endoplasmic reticulum. The orange label represents the site of follicle stimulating hormone (FSH) antigens. Thus, this cell contains FSH antigens and has transcribed GH mRNA. These are the same multipotential growth hormone cells seen just before ovulation that augment the gonadotrope population.
For more information, see the Growth
hormone Web page
Electron micrograph showing labeling for Luteinizing hormone beta subunit mRNA on dilated rough endoplasmic reticulum (RER) in a pituitary gonadotrope.
| Solutions | |
| Hybridization buffer | |
| Prehybridization Protocol | |
| Post hybridization steps: detection of hybrids | |
| Detection of Peroxidase | |
| Dual-labeling for antigens | |
| Illustrations of dual-labeling | |
| Electron micrograph illustrating labeling on rough endoplasmic reticulum. | |
| Publications using these methods | |
| Vendors for in situ hybridization histochemistry. | |
| Important links to courses, protocols, and services in in situ hybridization. |
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The protocol is applied to cells plated for 1h -36 h on 13 mm glass coverslips ( Thomas Scientific , Catalog number 6672A75) in 24 well trays ( Fisher Scientific Catalog number 08-757-156). The protocol can also be used on frozen sections or paraffin sections. | |
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The probes used for the hybridization are either cRNA probes or complementary oligonucleotide probes at least 30 mer long. They have been biotinylated by the Vector Photobiotin kit ( Vector Laboratories,) Burlingame Calif), or with Biotin-UTP, during in vitro transcription. Most recently, they were produced by www.GeneDetect.com, with Greenstar ® technology. See this link for more information.All fixation, washing, and handling methods are run under sterile conditions to prevent RNase contamination. Controls include substitution of labeled sense sequences, or omission of the labeled probe. The protocol can be adapted for use with dispersed cells at the electron microscopic level. |
Solutions for in situ hybridization
| Phosphate buffered saline (PBS): 0.1M phosphate buffer + 0.9% NaCl pH 7.2 | |
| Triton X-100 (0.3%): 300 microliters Triton X-100 ( Sigma Chemical) & bring to 100 ml.with 0.1M PBS | |
| 50 mM EDTA : add 1.46 gm. EDTA to 100 ml. of 0.1M Tris
buffer ( Sigma Chemical), Catalog T-5030, (20 ml. 0.5M
Tris + 80 ml. Millipore-filtered water) pH 8.0
Diluent
for the proteinase K:
50 mM Tris with 2 mM Calcium chloride. | |
| 4% para-formaldehyde: 4.0 gm. p-formaldehyde in 100 ml.
0.1M PBS pH 7.2 0.25% acetic anhydride + 0.1M triethanolamine: 250 ul acetic anhydride & bring to 100 ml. millipore filtered water, then add 1.856 gm. triethanolamine, pH 8.0 ( Sigma Chemical) | |
| 50% formamide in 2X SSC: make a 1:10 dilution from 20X SSC with Millipore-filtered water and add 1:1 formamide ( Sigma Chemical), Catalog F-7503 | |
| 4X SSC : make a 1:5 dilution from 20X SSC with millipore
filtered water |
Preparation of In Situ Hybridization buffer :
The components come from Sigma Chemical , unless otherwise noted.
| Deionize formamide: add 1.5 gm. REXYN I-300 ( Fisher Scientific, Catalog number R-208) to 100 ml. formamide, stir for 45-60 min., filter thru Whatman filter paper | |||||||||||
| Combine 100 ml. deionized formamide + 100 ml. 4X SSC (20 ml. 20X SSC + 80 ml. millipore filtered water) | |||||||||||
| Add 7.88 gm. Tris salt---0.25M pH 7.5 , warm to about 37 degrees in 1.0 ml. millipore filtered water | |||||||||||
With sterile syringe & needle add the following to the warm
formamide-Tris solution:
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| finally, add 200 ul of dissolved salmon sperm DNA (ssDNA) to 200 ml. of hybridization buffer | |||||||||||
| aliquot into 10 ml. & store frozen (-20 C) in 15 ml.
centrifuge tubes |
Prehybridization Protocol
| Cells are grown on glass
coverslips ( Thomas Scientific , Catalog number 6672A75),
in Dulbeccos modified Eagles Medium ( JRH Biosciences ,
Catalog number 56499-10L), and fixed in 2.5% glutaraldehyde (in 0.1 M phosphate buffer)
for 30 min. Then cells are washed for 1 h in 4 changes of phosphate buffer + 4.5% sucrose.
Work under sterile conditions and store no longer than 1 wk at 2-4 C. | |
| rinse cells on coverlips with
fresh 0.1M Phosphate buffered saline (PBS)--5 mins. room temp., shaking. Then treat in the
following sequence of solutions. All of the following chemicals but the paraformaldehyde
came from Sigma Chemical | |
| treat with 0.3% Triton X-100---15 min. room temp. | |
| wash coverslips with 0.1M PBS---2X, 3 min. each, room temp, shaking | |
| treat with Proteinase K (in 50 mM Tris with 2 mM Calcium chloride.)---15 mins. room temp. | |
| postfix with 4% p-formaldehyde/0.1M PBS---5 mins, room temp. | |
| wash coverslips with 0.1M PBS---2X, 3 min. each room temp, shaking | |
| treat with 0.25% acetic anhydride---10 min. room temp, shaking | |
| treat with 50% formamide/2X SSC---10 min. room temp, shaking & then incubate at 37 degrees for 10 mins. | |
| hybridization step: incubate cells with 200 ng/ml. of
biotinylated oligonucleotide or cRNA-probe in hybridization buffer at 37 degrees overnite |
Post hybridization steps:
| wash coverslips with 4X SSC---3X, 15 min. each room temp. (gentle shaking during the last 5 mins.) | |
| block with 0.05M Tris buffered saline + 1% Bovine serum albumin + 10% Normal Horse Serum ( Vector Laboratories) ---15 min. room temp | |
| treat with monoclonal Anti-Biotin (1:30) ( Dako, Corp )---30 min. at 37 degrees | |
| wash coverslips with 0.05M Tris buffered saline---2X, 3 min. each | |
| incubate with Biotinylated Horse anti-mouse IgG (rat absorbed, Vector Laboratories) | |
| 1:100---10 min. room temperature | |
| wash coverslips with 0.05M Tris Buffered saline---2X, 3 min. each | |
| incubate with second layer of monoclonal Anti-Biotin (1:100)---30 min. at 37 degrees | |
| wash coverslips with 0.05 M Tris buffered saline---2X, 3 min. each | |
| incubate with second layer of Biotinylated horse anti-mouse IgG (rat absorbed) 1:100 ---10 min. room temperature | |
| wash coverslips with 0.05M Tris buffered saline---2X, 3 min. each | |
| incubate with 1:10 peroxidase conjugated streptavidin ( Dako, Corp, Catalog number P-397) ---5 min. Room temperature | |
| wash coverslips with 0.05M Tris buffered saline---2X |
Detection of peroxidase:
| prepare nickel diaminobenzidine---dissolve 0.45 gm. nickel ammonium sulfate in 30 ml. 0.05M acetate buffer, add 1 Diaminobenzidine tablet ( Sigma Chemical , Catalog number D-5905). Dissolve and then add 20 microliters of 30% hydrogen peroxide. Filter. Add to cells for 6 min. Reaction product is blue-black | |
|
wash coverslips with 0.05M acetate buffer---2X;-dehydrate, dry
& permount. Note: This reaction product is dissolved in water soluble mounting media
or glycerol. Therefore, use only organic solvent soluble mounting media with it. |
After the mRNA is detected with the blue-black peroxidase substrate, the cell can be further labeled by immunocytochemistry for its protein or antigen content. For this we use the dual-labeling protocol with contrasting color peroxidase substrates.
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 pituitary antigen. This allows us to identify the cell that contains the mRNA by its antigen content. The above photo shows the dual reaction for LH antigens (orange) and GH mRNA (arrows) in gonadotropes from proestrous rats. The following outline shows an example of the protocol that detects the second antigen (such as luteinizing hormone or follicle stimulating hormone, or growth hormone). This allow us to identify the hormone content of the cell that binds the ligand, expresses the mRNA or expresses more than one hormone. For more information, see the GH web site.
To detect antigens after the detection of the mRNA:
| After in situ hybridization is completed, rinse coverslips with 0.05M Tris buffered saline---1X | |
| block again with 0.05M Tris buffered saline + 1% Bovine serum albumin ( Sigma Chemical, Catalog number A-7638)---15 min. room temperature | |
| incubate with either 1:30K Anti-LH/1:10K Anti-FSH---30 min. at 37 degrees | |
| wash coverslips with 0.05M Tris buffered saline---3X | |
| incubate with Biotinylated Goat anti-rabbit IgG ( Vector Laboratoriesl, Catalog number BA-1000) ( 25 microliters stock Biotinylated-IgG + 25 microliters Normal goat serum in 2 milliliters of buffer )---20 min., 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 min. 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 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 min. 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 the slides is to dehydrate the tissues and use permount. |
Publications using these methods
Use of the Photobiotinylated probe and the ABC-peroxidase complex for detection of the probe
| Childs, G.V., Lloyd, J., Unabia, G., Gharib, S.D., Wierman, M.E. and Chin, W.W. Detection of LH mRNA in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated cRNA probe. Molecular Endocrinology 1:926-932, 1987. | |
| Wu, Ping and Childs, G.V. Cold and Novel environment stress affects AVP mRNA in the paraventricular nucleus, but not the supraoptic nucleus: an in situ hybridization study. Molecular and Cellular Neurosciences, 1:233-249, 1990. | |
| Childs, G.V., Patterson, J., Unabia, G., Rougeau, D. and Wu, P. Epidermal growth factor enhances ACTH secretion and expression of POMC mRNA by corticotropes in mixed and enriched cultures. Molecular and Cellular Neurosciences, 2: 235-243 1991. | |
| Wu, Ping A. and Childs, G.V. Changes in rat pituitary POMC mRNA after exposure to cold or a novel environment detected by in situ hybridization. Journal Histochem Cytochem. 39(6):843-852, 1991. | |
| Childs, G.V., Taub, K., Jones, K.E. and Chin, W.W. Tri-ioodothyronine receptor -2 mRNA expression by somatotropes and thyrotropes: Effect of propylthiouracil-induced hypothyroidism in rats. Endocrinol, 129:2767-2773, 1991. | |
| Childs, G.V., Unabia, G., Lloyd, J. Recruitment and maturation of small subsets of luteinizing hormone (LH) gonadotropes during the estrous cycle, Endocrinology, 130:335-345 1992. | |
| Childs, G.V. Unabia, G. Lloyd. J.M Maturation of FSH gonadotropes during the rat estrous cycle. Endocrinology 131(1):29-36, 1992. | |
| Childs, G.V. , Unabia G., Rougeau D. Cells that Express Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) Beta ( ) Subunit mRNAs during the Estrous Cycle: The major contributors contain LH , FSH and/or Growth Hormone, Endocrinology, 134: 990-997 1994. | |
| Kaiser, U, Lee, BL, Unabia, G, Chin, W, Childs, G.V. Follistatin gene expression in gonadotropes and folliculostellate cells of diestrous rats. Endocrinology 130(5):3048-3056, 1992. | |
| Lee, B.L., Unabia, G., Childs, G. Expression of follistatin mRNA in somatotropes and mammotropes early in the estrous cycle J. Histochem. Cytochem, 41: 955-960, 1993. | |
| Fan, X. and Childs GV 1995. EGF and TGF mRNA and their Receptors in the Rat Anterior pituitary: Localization and Regulation; Endocrinology, 136: 2284-2324. | |
| Armstrong J and Childs GV 1997 Changes in expression of epidermal growth factor receptors by anterior pituitary cells during the estrous cycle. Endocrinology 138: 1903-1908 | |
| Childs, GV Unabia, G, and Wu, P. Differential expression of growth hormone messenger ribonucleic acid by somatotropes and gonadotropes in male and cycling female rats. Endocrinology 141: 1560-1570, April, 2000 | |
Childs GV Simultaneous identification of a specific gene protein product and transcript using combined immunocytochemistry and in situ hybridization with non-radioactive probes. Scanning Microscopy International, Supplement 10 pp 17-26. | |
| Childs, GV In situ hybridization with non-radioactive probes, Methods in Molecular Biology, Humana Press, Totowa, NJ, pp131-141, 1999 | |
| Childs, GV In situ hybridization, In Genetics, an encyclopedia. GaleGroup Inc., NY. In press | |
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McDuffie, I, Akhter, N, and Childs, GV Regulation of leptin expression in anterior pituitary somatotropes. J Histochem Cytochem, 52: 263--273 (2004). | |
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Childs GV, Iruthayanathan M, Akhter N, Unabia G, Whitehead-Johnson B. 2004 Bipotential Effects of Estrogen on Growth Hormone Synthesis and Storage, in vitro Endocrinology Apr;146(4):1780-8. | |
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Iruthayanathan, M., Zhou, Y, and Childs, GV 2005 DHEA Restoration of GH Gene Expression in Aging Female Rats, in vivo and in vitro - Evidence for Actions Via Estrogen Receptors. Endocrinology, epub ahead of print, September 8, 2005; 146(12):5176-87. |
Electron microscopic study:
| Childs, G.V., Unabia, G., Weirman, M.E., Gharib, S.D. and Chin, W.W. Castration induces time-dependent changes in the FSH -mRNA-containing gonadotrope cell population. Endocrinology 126:2205-2213, 1990. | |
| Use of a cRNA probe biotinylated by in vitro transcription and detected by the 5 step immunolabeling method (as described in this Web page) |
Vendors for Cytochemistry Techniques
| GeneDetect for Oligoprobes. For reliable, highly sensitive anti-sense oligonucleotide probes, we use www.GeneDetect.com. This company uses a variety of labeling systems and we have had excellent success with their biotinylated and digoxygenin-labeled oligonucleotide probes for GH, LH, FSH, and leptin mRNA. They will also design the probes and provide support. Each probe comes with a kit that includes labeled sense and antisense sequences as well as unlabeled sequences for competition experiments. They respond rapidly to all requests. | |
| Dako Corporation, 6392 Via Real, Carpenteria, Ca 93013; 800-235-5763. | |
| 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, Swedesboro, NJ 08085-9904 800-345-2100 | |
| Vector Laboratories, 30 Ingold Road, Burlingame, Ca 94010, 800-227-6666.
Web page: Vector
Laboratories |