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
We are constructing a page that describes the dual-in
situ protocol for biotinylated and digoxygenin labeled probes.
For more information on these protocols, see
the Journal of Histochemistry and
Cytochemistry web site.
What's new at this site: March 2006
The figure shows cells labeled with
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
micrograph showing labeling for Luteinizing hormone beta subunit mRNA on dilated rough
endoplasmic reticulum (RER) in a pituitary gonadotrope.
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.
The probes used for the hybridization are
either cRNA probes or complementary oligonucleotide probes at least 30 mer long. They
biotinylated by the Vector Photobiotin kit ( Vector
Laboratories,) Burlingame Calif), or with Biotin-UTP, during in vitro
transcription. Most recently, they were produced by
See this link for more
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
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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
0.5 gm. bovine serum albumin (RIA grade) ( Sigma Chemical, Catalog #A-7638)
0.5 gm. Ficoll-400
0.5 gm. PVP-360
1.0 gm. sodium pyrrophosphate
1.0 gm. SDS (lauryl sulfate
sodium salt) |
| 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.
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|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,
| treat with Proteinase
50 mM Tris with 2 mM Calcium chloride.)---15 mins.
| postfix with 4% p-formaldehyde/0.1M PBS---5 mins, room temp.
| wash coverslips with 0.1M PBS---2X, 3 min. each room temp,
| 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|
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| 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.
| 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.
| 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|
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Detection of peroxidase:
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.
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.
| incubate with either 1:30K Anti-LH/1:10K Anti-FSH---30 min. at 37
| 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)
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. |
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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,
| 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,
| 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
|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,
Childs, GV In situ
hybridization, In Genetics, an encyclopedia. GaleGroup Inc., NY. In
McDuffie, I, Akhter, N, and
Childs, GV Regulation of leptin expression in anterior pituitary
somatotropes. J Histochem Cytochem, 52: 263--273 (2004).
Childs GV, Iruthayanathan M,
Akhter N, Unabia G, Whitehead-Johnson B. 2004 Bipotential Effects of
Estrogen on Growth Hormone Synthesis and Storage, in vitro
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;
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)
for Cytochemistry Techniques
GeneDetect for Oligoprobes.
For reliable, highly sensitive anti-sense oligonucleotide probes, we use
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.
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
Biosciences, PO Box 14848 Lenexa, KS 66325 800-255-6032
| Sigma Chemical, PO Box 14508; St. Louis, MO 63178;
Link to their Web page at Sigma
Chemical Web page .
Scientific, 99 High Hill Road, PO Box 99, Swedesboro, NJ
| Vector Laboratories, 30 Ingold Road, Burlingame, Ca 94010, 800-227-6666.
Web page: Vector