In Situ
Hybridization of Mucin mRNA 323
323
27
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.
In Situ
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.)
In Situ
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.
328 Gipson
2. 0.2 M EDTA, pH 5.2.
3. 3 M Na acetate, pH 5.2.
4. tRNA.
5. 75% ETOH.
2.5. Prehybridization Solutions
1. Phosphate-buffered saline (PBS), pH 7.4.
2. Proteinase K.
3. 100 mM Tris-HCl, pH 7.6.
4. 0.5 M EDTA, pH 7.5.
5. 0.2% Glycine in PBS.
6. 4% Paraformaldehyde in PBS.
7. 1 M Triethanolamine, pH 8.0.
8. Acetic anhydride.
9. 20X Sodium chloride/sodium citrate (SSC) buffer: 3 M NaCl, 0.3 M sodium citrate; adjust
pH to 7.0 with 1 M HCl.
2.6. Hybridization Solutions and Supplies
1. Formamide (Sigma, St. Louis, MO).
2. 10X salt buffer: 3 M NaCl, 0.1 M Tris-HCl, pH 7.6, 50 mM EDTA, 0.2% Ficoll 400, 0.2%
polyvinylpyrrolidone, 0.2% BSA.
3. 1 M Dithiothreitol (DTT) (not necessary for immunodetection method).
4.
35
S or DIG-labeled sense and antisense oligonucleotide probes.
5. 50% Dextran sulfate.
6. tRNA.
7. Cover Wells™, or Probe Clips
®
, which are cover slips with sealing gaskets that provide
moist, well-sealed chambers for the hybridization step (available from GBL, Pontiac, MI,
or PGC Scientific, Frederick, MD).
8. Slide holder (Sigma Humid Chamber, cat. no. 6644).
9. Sealable moist plastic box.
2.7. Posthybridization Solutions
1. 20X SSC.
2. Formamide.
3. 14 M β-mercaptoethanol.
4. Ribonuclease (i.e., RNase) (Boehringer Mannheim).
2.8. Autoradiography/Counterstaining
for Disclosure of
35
S Oligonucleotide Binding
1. Kodak NTB2 Autoradiography Emulsion (cat. no. 165 4433, Kodak, Rochester, NY).
2. Light-tight black box.
3. Kodak D19.
4. Kodak fixer.
5. Hematoxylin and eosin (H&E) stain.
2.9. Disclosure of DIG-Labeled Oligonucleotide Probe
2.9.1. Colorimetric-Alkaline Phosphatase
Kits are available from Boehringer Mannheim.
In Situ
Hybridization of Mucin mRNA 329
1. Buffer 1: 0.1 M Tris-HCl, 0.15 M NaCl, pH 7.5.
2. 1% Dry milk in buffer 1; alternate 1% BSA in buffer 1.
3. Anti-DIG-alkaline phosphatase conjugate: sheep anti-DIG, Fab fragments, conjugated
with alkaline phosphatase, 750 U/mL.
4. Alkaline reaction buffer: 0.1 M Tris, 0.1 M NaCl, 50 mM MgCl
2
, 0.1% Tween-20, pH 9.5.
5. Nitroblue tetrazolium (NBT) salt.
6. 5-bromo-4-chloro-3-indolyl phosphate toluidinium (BCIP) salt.
2.9.2. Fluorescent ISH (FISH)
1. Buffer 1: 0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5.
2. 1% Dry milk in buffer 1.
3. Anti-DIG-rhodamine-Fab fragments (Boehringer Mannheim) with a final concentration
of 20 µg/mL.
3. Methods
3.1. Preparation of Tissue
1. To prepare fixative (4% paraformaldehyde in RNase-free 0.1 M phosphate buffer, pH
7.4), heat buffer to 60˚C and add 4 g of paraformaldehyde/100 mL of buffer. Add 1–3
drops of 1 N NaOH. Store at 4˚C for up to 1 mo.
2. Fix tissue for 1–24 h at room temperature. Ideally, tissues are fixed immediately or within
1 h of biopsy or death (see Note 2).
3. Rinse tissue in RNase-free 0.1 M of phosphate buffer, pH 7.4 (three times for 15 min),
dehydrate in ETOH series followed by xylene, and embed in paraffin, using standard but
RNase-free techniques. Store blocks at 4˚C.
3.2. Preparation of Slides
Prepare gelatin-coated slides. In our experience, gelatin is superior to other “sub-
bing” compounds in that loss of tissue sections during the hybridization procedure is
minimal (14). For “subbing slides”:
1. Transfer slides to metal carrier and soak in 100% ETOH overnight in glass dishes.
2. Discard ETOH and bake slides in glass dishes for 2 h at 180°C.
3. Cool at room temperature.
4. Dip in 1% gelatin/0.1% chromium potassium sulfate solution for 10 min, and then
allow to dry. The gelatin solution is made by dissolving 3 g of gelatin in 200 mL: of
DEPC-treated water, which is warmed to 60°C until gelatin is completely dissolved.
Separately, 0.3 g of chromium potassium sulfate is added to 100 mL of DEPC-
treated water and mixed at room temperature until dissolved. The two solutions are
combined.
5. Fix slides in 4% paraformaldehyde in 0.1 M phosphate buffer for 15 min.
6. Wash in DEPC-treated distilled water two times for 5 min each.
7. Dry and store at room temperature in a clean box.
3.3. Sectioning of Tissue
1. Section paraffin-embedded tissue at 6 µm, mount on subbed slides, and store slides at 4°C
until use.
2. Heat slides to 40°C overnight just prior to use.
330 Gipson
3.4. Preparation and Labeling of Oligonucleotide Probes
Oligonucleotide probes and riboprobes (see Note 3) have been used for radioiso-
tope or immunodetection of mucin mRNA in tissues. Table 1 lists examples of probes
used in ISH studies of mucin gene expression. Oligonucleotides to mucin TR sequence
work quite well and provide the simplest method for labeling. Riboprobes provide a
more sensitive method of message detection and are useful for quantitative assays
when using non-TR probes. For examples of
35
S-labeled oligonucleotide and riboprobe
as well as DIG-alkaline phosphatase or FISH, see Figs. 1 and 2.
3.4.1. Labeling of Oligoprobes with
35
S
1. Mix the following in a microfuge tube on ice:
a. 4 µL of 5X reaction buffer.
b. 10 pmol of oligonucleotide probe.
c. 2.5 µL of
35
S-dATP.
d. 1 µL of terminal deoxynucleotidyl transferase (TdT).
e. Water to a final volume of 20 µL.
2. Incubate at 37°C for 1 h and microfuge for 1–2 min.
3. Add 4 µL of 0.2 M EDTA to terminate the reaction on probe purification (see Note 4).
4. To precipitate the probe, add 0.1 vol of 3 M Na acetate, pH 5.2, 2.5 vol of alcohol, and 0.2
vol of 1 mg/mL tRNA in DEPC-treated water and store at –80°C for 2 to 3 h or overnight.
5. Centrifuge the probe at 12,000g (15–20 min), wash the pellet with 50 µL of cold ethanol
(75%, v/v), and air- or vacuum-dry the pellet.
6. Dissolve the pellet in 10 µL of DEPC-treated water.
7. Use 1 µL of the probe to check counts per minute in scintillation counter.
8. Store probe at –80°C.
3.4.2. Labeling of Oligonucleotide Probe with DIG-UTP (
see
Note 5)
1. Mix well the following in an RNase-free microfuge tube on ice:
a. 10 pmol of probe.
b. 4 µL of 5X reaction buffer.
c. 4 µL of 25 mM CoCl
2
.
d. 1 µL of 1 mM DIG-dUTP solution.
e. 1 µL of 10 mM dATP.
f. 1 µL of TdT.
g. Water to final volume of 20 µL.
2. Incubate at 37°C for 1 h.
3. Add 2 µL of stop solution (mixture of 1 µL of glycogen and 200 µL of 0.2 M EDTA) to
stop the reaction. (See Note 4 on NucTrap Column.)
4. Precipitate the labeled oligonucleotide probe with 0.1 vol of 3 M Na acetate, pH 5.2, 2.5
vol of ETOH, and 0.2 vol of 1 mg/mL tRNA at –80°C for 2 h or overnight.
5. Centrifuge the probe at 12,000g (15–20 min), wash the pellet with 50 µL of cold ethanol
(75%, v/v), and air- or vacuum-dry the pellet.
6. Dissolve the pellet in 10 µL of DEPC-treated water.
7. Store labeled oligonucleotide at –80°C for up to 1 yr.
3.5. Prehybridization, Proteinase K Treatment, and Acetylation
1. Select enough slides so that both antisense and sense probes may be used (see Note 6).
You may put in an extra slide for quick X-ray film assay to determine the success of
labeling (see Note 7). Incubate at 40°C overnight.
In Situ
Hybridization of Mucin mRNA 331
2. Deparaffinize slides in the following:
a. Xylene for 10 min (two times).
b. 100% ETOH for 4 min.
c. 90% ETOH for 4 min.
d. 75% ETOH for 4 min.
3. Fix in 4% paraformaldehyde in PBS for 10 min.
4. Rinse in PBS for 3 min.
5. Treat with proteinase K to increase accessibility of probe to mRNA in fixed tissue. Stock:
2.5 mg/mL in 10 mM Tris-HCl, pH 7.6. Warm proteinase solution to 37°C before adding
slides.
a. 10 mM Tris-HCl/1 mM EDTA (TE buffer) for 5 min.
b. 1 µg/mL ofproteinase K in TE buffer for 20 min at 37°C.
c. 0.2% Glycine in PBS for 5 min.
d. PBS for 3 min.
e. 4% Paraformaldehyde in PBS for 20 min.
f. PBS for 5 min.
6. Treat with acetic anhydride to block nonspecific binding. Important: Add acetic anhy-
dride to triethanolamine just prior to treating slides.
Table 1
Examples of Probes and Disclosure Methods for
In Situ
Hybridization
to Localize Mucin mRNAs
Mucin gene Probe designation/type Probe length (bp) Disclosure used Refs.
MUC1 MUC1-1/2 450
35
S (7,10,20)
Oligo 48
35
S (21)
MUC2 HAM-1 92
35
S (20–22)
SMUC41 836 DIG (22,23)
Oligo 48
35
S (24)
Muc3 Riboprobe 473
35
S (11)
Oligo 48
35
S (24)
MUC4 Oligo 48
35
S (24)
Oligo 48 DIG/FISH (9)
Oligo 48 DIG/colorimetic (8)
MUC5AC Oligo 48
35
S (24)
4F 494
35
S (8)
4F 494 DIG/FISH (9)
PM5 1300 DIG/colorimetric (25)
MUC5B ngBM4-1 984
35
S (10,26)
Oligo 48
35
S (21,24)
MUC6 Oligo 30 DIG/colorimetric (27)
Oligo 39
35
S (10)
Oligo 74
35
S (28)
Oligo 48 DIG/colorimetric (29)
PM6 840 DIG/colorimetric (25)
MUC7 Oligo 48
33
P (30)
Oligo 48 DIG/colorimetric (27)
332 Gipson
a. 0.1 M Triethanolamine, pH 8.0, containing 1/200 vol/vol of acetic anhydride for 10 min.
7. Store slides in 2X SSC until hybridization solution is ready.
3.6. Hybridization
1. Make hybridization buffer (need 200 µl/slide), i.e., add:
a. 1200 µL of formamide.
b. 480 µL of 50% dextran sulfate.
c. 24 µL (leave out for DIG method) of 1 M DTT.
d. 240 µL of 10X salt buffer.
e. 50 µL of 1 mg/mL tRNA.
f. DEPC-treated water to a final volume of 2400 µL.
2. Heat hybridization buffer at 80°C for 5 min and mix well.
3. To make hybridization solution, add probe to buffer to a final concentration of 5 × 10
3
cpm/µL for
35
S-labeled oligoprobe or to an amount of 0.5–1 µg/mL of DIG-labeled probe.
4. Take slides out of SSC and air-dry.
5. Add 200 µL of hybridization solution to each slide and cover with a 200 µL-Probe Clip or
cover well to seal.
6. Place flat on to a slide holder and then into a sealed moist chamber (plastic box), and
place in a 37°C-oven overnight.
3.7. Posthybridization Washes (
see
Note 8)
1. Dip slides once in 2X SSC to remove Probe Clip or cover well. Then wash as follows:
a. 2X SSC at room temperature for 30 min.
b. 1X SSC at room temperature for 30 min.
c. 0.5X SSC at 37°C for 30 min.
d. 0.5X SSC at room temperature for 30 min.
2. For disclosure by autoradiography, dry slides overnight; for disclosure by the immuno-
detection method, enter the slides into the detection protocol after the last SSC wash.
3.8. Autoradiographic Detection of Probe
1. Warm water bath to 43˚C to melt emulsion.
2. Use Kodak NTB2 Autoradiography Emulsion diluted 1:1 with warm water. Use emulsion
under safelight or in complete darkness.
3. Dip slides, one at a time, in the emulsion and stand to dry for 2 h. This should be done
under a dim safelight or in complete darkness. Always dip a completely blank slide as a
negative control for the quality of the autoradiography for each condition/development time.
4. Store slides in a light-tight black box with desiccant, taped shut in a black plastic enve-
lope at 4˚C for 1–4 wk.
5. Develop slides as follows:
a. Allow slides to come to room temperature before developing (~30 min).
b. Develop in Kodak D-19 1:1 dilution with water for 5 min room temperature.
c. Stop in distilled water and briefly rinse.
d. Fix in full-strength Kodak Fixer for 15 min at room temperature.
e. Check slides under safelight to make sure fixation is complete (slides are clear, not opaque).
6. Wash in gently flowing water for 30–60 min.
7. Lightly counterstain in H&E.
8. Dehydrate through ethanols to xylene and cover slip with Permount (Fisher Scientific,
Pittsburgh, PA).
In Situ
Hybridization of Mucin mRNA 333
3.9. Immunodetection of Probe
3.9.1. Colorimetric–Alkaline Phosphatase Detection of Binding of DIG-
Labeled Oligonucleotide Probe to Tissue Sections
1. Wash slides in buffer 1 for 10 min.
2. Block in 1% dry milk in buffer at room temperature, 30 min.
3. Incubate with 1/500 anti-DIG-alkaline phosphatase conjugate at room temperature for 2 h
or overnight at 4°C.
4. Wash with buffer 1 two times for 15 min each.
5. Equilibrate the slides with alkaline reaction buffer for 2 min.
6. Develop the color with color-substrate solution: 1:50 dilution of NBT/BCIP stock from
Boehringer Mannheim (45 µL of NBT solution and 35 µL of BCIP solution in 10 mL of
buffer 1 at room temperature).
7. Stop the reaction with distilled water and wash twice.
8. Mount with Vectashield and a cover slip.
3.9.2. Fluorescent Antibody Detection of Binding of DIG-Labeled
Oligonucleotide Probes to Tissue Sections
1. Wash slides in buffer 1 for 5 min.
2. Block in 1% dry milk in buffer 1 for 30 min.
3. Incubate with 20 µg/mL of anti-DIG-rhodamine at 37˚C for 1 h in a sealed moist chamber.
4. Wash in buffer 1 three times for 10 min each.
5. Mount sections with mounting media.
4. Notes
1. For general methods, glassware baking, and DEPC treatment of water, bake all glassware
slides and heat-resistant equipment for 2 h at 180°C to make RNase free. To make RNase-
free water and buffers, add 0.1% DEPC, stir for 10 min, let sit overnight, and autoclave
the following day.
2. Fixation time may depend on the size of the excised tissue. Reports vary on overfixation (14–
16): Some investigators suggest that overfixation causes excessive crosslinking that prevents
probe access, whereas others suggest that it induces nonspecific binding. Underfixation in the
center of tissues of a large block is cited as another potential problem (14). All agree that
fixation of tissue as soon as possible after death or biopsy is key (<30 min).
3. Riboprobes (RNA probes) are currently considered the gold standard of ISH because they
provide a more sensitive assay. This is owing in part to the fact that the riboprobe is
labeled with the disclosing agent, be it radioisotope or DIG, as it is transcribed from the
cDNA template. Labeled nucleotide is incorporated all along the RNA sequence; thus,
the probe is heavily labeled, allowing an enhanced signal. Riboprobes are especially suit-
able for tissues in which there are low copy numbers of the mRNA in question or if mucin
probes to be used are not TR probes (suitable for quantitative assay). The preparation of
the riboprobe is labor-intensive. One must derive the riboprobe from appropriate tem-
plate cDNA. The template cDNA of ideally 100–300 bp may be obtained in several ways:
(1) elution from an agarose gel of a polymerase chain reaction product obtained by
designing primers for a known sequence of a mucin molecule from RNA or DNA of
tissue known to express the mucin; (2) excision of a smaller fragment from a large cDNA
by endonucleases; (3) in a plasmid vector, from a colleague. After the cDNA template is
obtained, the following steps are required for riboprobe production and labeling. The
334 Gipson
template cDNA is ligated into an appropriate plasmid vector (i.e., Bluescript), the plas-
mid vector is transfected into Escherichia coli, and positive colonies isolated. Positive
colonies are amplified, plasmid DNA is purified from E. coli cultures, and the presence
and direction (to determine sense, antisense sequence) of inserted template cDNA is veri-
fied and determined, respectively. The plasmid is then linearized and reverse transcribed
with polymerases, with the choice depending on vector; the common vector Bluescript
uses SP6, T7, or T3 with simultaneous labeling with either
35
S-UTP or DIG-UTP, result-
ing in a labeled riboprobe ready for ISH. For methods of template cDNA, riboprobe pro-
duction, and labeling, the reader is referred to (6,16).
4. One alternate method of removing unincorporated nucleotides is the use of a Sephadex
G-25 column (5 × 0.5 cm). Equilibrate the column with buffer (10 mM Tris-HCl, pH 7.5,
1 mM EDTA, 0.1 M NaCl), add labeled probe mixture, incubate, and elute. Unbound free
nucleotides remain in the column. A second alternate and convenient method is the use of
commercially available “push column” devices (i.e., NucTrap Push Columns, Stratagene
Cloning Systems, cat. no. 400701, La Jolla, CA).
5. For direct/indirect FISH, one can also use fluorescently labeled DIG to label the
oligoprobe. In our hands, we obtained enhanced signal using the indirect detection
method, that of fluorescently labeled anti-DIG antibody localization.
6. For slides, selections, and numbers, select a minimum of three slides of each tissue
tested—one for sense, two for antisense. Add extra slides of control tissues (e.g., epithe-
lia known to express the mucin gene in question), and several sets of slides for autorad-
iography, so that several exposure times may be tested (e.g., 1 and 2 wk).
7. For X-ray film preexposure, one can obtain some indication of strength of signal and
gross distribution by exposing slides to fast X-ray film. For
35
S, 2–5 d of exposure may be
required. For complete protocol, see ref. 16. With mucin mRNA localization, 1 wk is
often sufficient exposure for autoradiographic slides. Thus, the intermediate X-ray film
method may not be labor saving.
8. The posthybridization conditions of stringency (highest stringency equals lowest SSC
concentration) and temperature can be manipulated to remove nonspecific, less specifi-
cally associated oligoprobes so as to improve specific signal. Conditions must be empiri-
cally determined for each probe.
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