Glycoprotein methods protocols - biotechnology 048-9-323-336.pdf

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In Situ
Hybridization Techniques for Localizing Mucin mRNA
Ilene K. Gipson
1. Introduction
Progress in understanding how mucosal surfaces are protected is closely related to
the development of morphologic techniques to study the structure and secretory func-
tion of the mucosal epithelia. Morphologic methods have allowed characterization of
mucus-secreting cells of the epithelia of the eye, and the respiratory, gastrointestinal
(GI), and reproductive tracts. Characteristics of the mucus-secreting cells of these tis-
sues vary, and many questions remain regarding special characteristics of mucus
present over the differing mucosal surfaces. Recent progress in cloning and character-
ization of mucin genes has facilitated the use of in situ hybridization (ISH) to begin to
characterize the mucin gene repertoires and specific functions of mucins expressed by
the various epithelia, either those covering mucosal surfaces or glandular epithelia
contributing to the mucous layer on the surface of the tissue. ISH has been a particu-
larly valuable method in this regard, since antibodies to specific mucin proteins are
often difficult to use on tissues or secretions without heroic methods to deglycosylate
in order to make protein epitopes available.
Mucins, because of their heavy glycosylation and size, have presented major tech-
nical difficulties to biochemists and molecular biologists struggling to characterize
them (1–3). The use of molecular techniques to sequence the mucin gene has identi-
fied a characteristic common to all mucin genes, that of tandemly repeated sequences
in their amino acid/nucleotide sequence. (For review see refs. 4 and 5). This character
greatly facilitates application of ISH methods to localize specific mucin mRNAs in
tissues and cells. Probes to the tandemly repeated nucleotide sequences bind at mul-
tiple sites along cellular mRNA, providing an amplified signal and excellent visual-
ization of the presence of specific mucin mRNAs. For once, there is something about

mucin character that facilitates ease of application of a method! While this enhanced
signal is useful, it is an impediment to quantitative assays. One cannot rely on the use
of tandem repeat (TR) probes to quantitate mRNA levels, especially with those mucin
genes that exhibit polymorphisms.
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
324 Gipson
Currently, the two probes of choice for ISH are riboprobes of usually 100–300 bp
(RNA transcribed from cDNA probes) or oligonucleotide probes of 18–100 bp (which
match the cDNA sequence), the latter being less sensitive. Because of the enhanced
signal obtained with TR probes, straightforward simple in situ methods can be applied.
One can thus employ less sensitive, labeled oligonucleotide probes with radioisotope
detection or with nonradioisotope immunodetection methods; the latter disclosure
method is also less sensitive. Special efforts to preserve all RNA in the tissues, usually
a requirement for tissues with low levels of message, is not always necessary; thus,
archived, less stringently fixed and processed tissue sometimes can be used. Because
of its relative simplicity, our probe of choice for ISH of mucin mRNA is, therefore, the
oligonucleotide probe, but we usually use radioisotope labeling at least in initial
experiments until we determine signal levels.
ISH methods have been applied to the study of mucin genes in two ways: (1) for
chromosomal localization of specific mucin genes, and (2) for tissue or cellular local-
ization of specific mucin mRNAs. This chapter describes protocols for tissue localiza-
tion only; for chromosome localization methodologies, readers are referred to ref. 6.
The methods described in this chapter are those that have been successfully applied in
our laboratory to determine specific mucin mRNA localization in epithelia covering
the eye, reproductive tract, and GI tract (7–11). Since the signal for mucin message is
usually easily detected in tissues, one does not have to be as concerned with loss of
low-level message and access to message. Thus, one can use paraformaldehyde-fixed,
paraffin-embedded tissue rather than frozen tissue and benefit from the better preser-

vation of tissue architecture.
Both radioisotope (
35
S) and immunodetection (digoxygenin [DIG]) methods of ISH
(colorimetric and fluorescence disclosure) are described, and both methods work well
with routine mucin mRNA localization. Of the methods described, the most sensitive
is that of the radioisotope labeling of probes. The colorimetric DIG protocol is useful
if one chooses not to use radioisotope methods, is not equipped for the work, or does
not have access to dark-field microscopy. It has the disadvantage that with colorimet-
ric disclosure methods, interpretation can be difficult to distinguish in counterstained
tissues with low expression levels. The fluorescent DIG ISH method gives the best
resolution of message within the cytoplasm of cells, especially when viewed with con-
focal microscopy. In our hands, however, this method is the most capricious of the
three disclosure methods and does not provide a permanent record. Figures 1 and 2
show examples of several methods of ISH as applied to mucin mRNA localization.
The protocols that follow are described in a rather practical and simple fashion. For
complete descriptions of the theory and practice of ISH, readers are referred to refs. 12–17.
2. Materials
All materials and solutions are prepared RNase free. Baked glassware is used and
all materials and equipment are handled with latex gloves. All water and buffers are
made RNase free by diethylpyrocarbonate (DEPC) treatment (see Note 1).
2.1. Equipment
1. Microtome.
2. Water bath: 30–60°C.
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Hybridization of Mucin mRNA 325
3. Microfuge.
4. Vortex mixer.
5. Oven/incubator: 30–60°C.
6. Heat block/water bath adjustable to 80°C.

7. Water bath (42°C) for autoradiography.
8. Light-tight darkroom.
2.2. Fixation and Embedding of Tissue in Paraffin
1. 4% Paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.
2. 0.1 M phosphate buffer, pH 7.4.
3. 100, 95, 70, and 50% ETOH.
Fig. 1. Micrographs demonstrating two methods of disclosure of oligonucleotide probes
binding to mucin mRNA. (A, C) Dark field; (B) and (D) H&E of the same field of conjunctival
epithelium, respectively. In (A),
35
S-labeled oligonucleotide (48 mer) to MUC4 TR sequence is
localized in all cell layers of the stratified epithelium. (B) is the sense control of (A); (E) is
antisense, and (F) is sense control of DIG-labeled MUC4 oligonucleotide probe disclosed with
alkaline phosphatase/NBT. Bars = 50 µm. (Reproduced by permission from ref. 8.)
326 Gipson
Fig. 2. Example of three methods of ISH using mucin mRNA probes on sections of human con-
junctiva. (A, B) Localization of MUC4 mRNA using antisense (A) and sense (B) oligonucleotide
probes labeled with DIG and disclosed with fluorescently labeled anti-DIG. Note MUC4 message
surrounds the nuclei of all the cells in the epithelium (A). (C–E) Use of riboprobes to localize MUC5AC
in goblet cells of human conjunctival epithelium. In (C) the riboprobe was labeled with DIG-labeled
UTP and the DIG was disclosed with fluorescently labeled anti-DIG. In (E), the riboprobe was labeled
with
35
S UTP and disclosed by autoradiography. Note that 5AC mRNA is restricted to goblet cells. (D)
and (F) are sense controls for (C) and (E), respectively. Bars = 20 µm. (Reproduced from ref. 9.)
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Hybridization of Mucin mRNA 327
4. Xylene.
5. Paraffin (e.g., Paraplast).
6. Embedding molds.

2.3. Preparation of Slides and Sectioning of Tissue
1. Microscope slides.
2. Gelatin.
3. Sodium potassium chromate.
4. 4% Paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.
5. Routine paraffin-sectioning supplies.
6. Coplin jars or glass staining dishes.
2.4. Preparation and Labeling of Oligonucleotide Probes
Synthesized oligonucleotides, both antisense and sense (>18 mer), appropriately
purified (16) are available from a variety of manufacturers. (For discussion of design
and synthesis, see refs. 17–19.)
Commercially available 3'-labeling (tailing) kits are available for labeling
oligoprobes with either radionucleotides or DIG. The kits are convenient and can be
an economical method. Companies providing kits include Boehringer Mannheim
(Mannheim, Germany), Promega (Madison, WI), and Stratagene (La Jolla, CA).
2.4.1. Labeling of Oligoprobes with
35
S
Kits containing items 1 and 2 can be purchased; they usually also contain 5 mM
CoCl
2
included in the buffer.
1. 5X buffer: 1 M potassium cacodylate, 0.125 M Tris-HCl, 1.25 mg/mL bovine serum albu-
min (BSA), pH 6.6.
2. Terminal transferase.
3. 0.2 M EDTA, pH 5.2.
4. 3 M Na acetate, pH 5.2.
5. tRNA.
6. 75% ETOH.
2.4.2. Labeling with DIG

1. Kits for labeling oligonucleotides with DIG that contain the following:
a. 5X reaction buffer: 1 M potassium cacodylate, 0.125 M Tris-HCl, 1.25 mg/mL of
BSA, pH 6.6.
b. 25 mM CoCl
2
solution.
c. 1 mM DIG-deoxy uridine triphosphate (dUTP).
d. 10 mM deoxyadenosine triphosphate (dATP) in Tris buffer.
e. Terminal transferase: 50 U/µL in 0.2 M potassium cacodylate, 1 mM EDTA, 200 mM
KCl, 0.2 mg/mL of BSA, pH 6.5, 50% (v/v) glycerol.
f. Control oligonucleotide: unlabeled, 20 pmol/µL.
g. Control oligonucleotide: DIG-dUTP/dATP, tailed 2.5 pmol/µL.
h. 0.25 mg/mL of supercoiled pUC18 control DNA in 10 mM Tris-HCl, pH 7.6, 1 mM EDTA.
i. 20 mg/mL of glycogen solution.
j. DNA dilution buffer: 50 µg/mL of herring sperm DNA in 10 mM Tris-HCl, 1 mM
EDTA, pH 8.0.
k. 10 mg/mL of poly (A) solution.