Bernhard H.F. Weber and Thomas Langmann (eds.), Retinal Degeneration: Methods and Protocols, Methods in Molecular Biology,
vol. 935, DOI 10.1007/978-1-62703-080-9_15, © Springer Science+Business Media, LLC 2013
High-Throughput RNA In Situ Hybridization
in Mouse Retina
The introduction of large-scale gene expression pro fi ling studies has greatly increased the need to rapidly
obtain high-quality cellular expression patterns of genes found to exhibit differential expression. The use
of large-scale nonradioactive RNA in situ hybridization makes this possible, and greatly increases the
general usefulness of this data. Here, we describe protocols for parallel analysis of up to 50 different gene-
speci fi c probes in mouse retinal sections.
Key words: RNA , Gene expression , Cellular resolution , Hybridization , Digoxigenin , Riboprobe ,
Chromogenic , Retina , Photoreceptor , Development
RNA in situ hybridization ( 1, 2 ) can be used to characterize the
cellular expression pattern of any RNA species. By designing anti-
sense probes that can undergo complementary base pairing with a
target sequence of interest, one can readily design a probe that can
strongly and selectively bind to virtually any target RNA, whether
or not it codes for protein. The high stability of RNA–RNA hybrids
means that hybridization conditions can be made especially strin-
gent, thus resulting in both low background signal and high sensi-
tivity. Complementary RNA (cRNA) probes are typically generated
from linear DNA templates, using recombinant viral RNA poly-
merases to directly incorporate modi fi ed bases that contain a label
of choice to allow detection of probe–target hybrids. Radioactive
nucleotide triphosphates can be used to allow for direct detection
of bound probe. Alternatively, chemically modi fi ed bases can be
used, allowing indirect probe detection using immunodetection.
Critical improvements came with the use of bases conjugated to
the highly antigenic small molecule digoxigenin for probe labeling,
which allowed the use of highly speci fi c alkaline phosphatase-
conjugated antibodies for immunohistochemistry ( 2, 3 ) . These
modi fi cations improved the speci fi city and sensitivity of the proto-
col to the point where it could be used to analyze many different
probes in parallel. One of the most spectacular uses of this approach
has been the effort of the Allen Brain Atlas consortium to map the
expression of all annotated mouse genes in the adult brain ( 4 ) .
In the retina, large-scale in situ hybridization has been instru-
mental in analyzing the results of SAGE ( 5, 6 ) and microarray
( 7, 8 ) data obtained by pro fi ling different developmental stages, or
mutant animals exhibiting developmental defects or retinal degen-
eration. While global expression pro fi ling of this sort generates vast
amounts of data, it is very hard to interpret meaningfully unless
one also knows the cellular expression pattern of any differentially
expressed genes. Even in cases where isolated cell subtypes or even
individual cells are pro fi led ( 9, 10 ) , it is important to use a different
experimental approach to con fi rm the validity of any results
obtained. The regular structure of the retina enables the major cell
types that express a given gene to be identi fi ed on the basis of their
laminar position, making this data particularly useful. Since up to
50 different cRNA probes can be run in parallel by a single inves-
tigator, with results obtained within 3–5 days, it is now feasible to
rapidly sort through all of the most potentially interesting hits
obtained in such experiments.
1. DEPC-treated water: Add DEPC to 0.1% fi nal concentration
in MilliQ ddH 2 O. Shake solution to mix, and leave overnight
at room temperature. Inactivate DEPC by autoclaving (15–
25 min at 15 psi) prior to use (see Note 1 ).
2. DEPC-treated PBS, SSC, EDTA, LiCl: Prepare these as
described above for DEPC-treated water. Do not treat any
buffer containing amines (e.g., triethanolamine, Tris, etc.)
with DEPC (see Note 2 ).
1. 10× RNA polymerase buffer (Roche).
2. 10× DIG NTP mix (Roche).
3. RNAse-free ddH 2 O (not DEPC treated).
4. RNAse inhibitor (Roche).
5. RNA polymerase (T7, T3, or Sp6).
6. RNAse-free DNAse to degrade probe.
7. RNAse-free 1.5 ml Eppendorf tubes or RNAse-free 96-well
2.2. Probe Preparation
217 15 High-Throughput RNA In Situ Hybridization in Mouse Retina
8. DEPC-treated 3 M NaOAC.
9. RNAse-free 100% EtOH.
10. 70% EtOH prepared with DEPC ddH 2 O.
1. 4% Paraformaldehyde (PFA): 45 ml ddH 2 O, 4 g PFA.
To prepare, heat to 60–70°C, add 1 drop 10 N NaOH, stir to
dissolve. Once PFA has fully dissolved, add 5 ml 10× PBS,
pH 7.5. Sterile fi lter and store on ice. Use PFA solution on the
day of preparation. Alternatively, stocks of 20% PFA in water
(dissolve as above) can be prepared ahead of time. These can
be thawed, and brought to 4% in 1× PBS prior to use.
2. Hybridization buffer (50 ml volume): 25 ml 100% ultrapure
formamide, 12.5 ml 20× SSC, pH 6.0, 5 ml 50× Denhardt’s
solution, 250 m g/ml fi nal yeast tRNA in DEPC water (store
resuspended aliquots at −80°C), 500 m g/ml fi nal salmon sperm
DNA, DEPC ddH 2 O (to 50 ml).
3. 20× SSC, pH 6.0 (1 l): Add 175.9 g NaCl and 88.2 g
Na 3 (C 3 H 5 O(COO)·2H 2 O) to 800 ml of ddH 2 O. Adjust pH to
6.0 with concentrated HCl. Adjust volume to 1,000 ml fi nal.
4. 50× Denhardt’s solution (1 l): 900 ml ddH 2 O, 5 g Ficoll 400,
5 g polyvinylpyrolidone, 5 g BSA (Fraction V), adjust volume
to 1,000 ml with ddH 2 O. Filter through a 0.2 m m fi lter. Store
in aliquots at −20°C.
5. Protease K solution: Dissolve at 0.5 mg/ml in DEPC-treated
water. Freeze in aliquots and store at −20°C. Do not reuse
6. RNAse buffer: 0.5 M NaCl, 10 mM Tris pH 7.5, 5 mM
7. B1 buffer: 0.1 M Tris pH 7.5, 0.15 M NaCl.
8. B2 buffer: B1 + 5% heat-inactivated normal sheep serum (HISS).
Place serum in a water bath at 56°C, 30 min to heat inactivate.
Store HISS at −20°C in aliquots.
9. B3 buffer: 0.1 M Tris–Cl pH 9.5, 0.1 M NaCl, 50 mM
MgCl 2 .
Filter through a 0.45 m m fi lter (see Note 3 ).
10. B4 buffer: 3.375 m l/ml NBT (100 mg/ml in 70% dimethyl-
formamide), 3.5 m l/ml BCIP (50 mg/ml in ddH 2 O), 0.24 mg/
ml levamisole in B3 buffer.
11. Gelvatol mounting media: 21 g PVA, 42 ml, 52 ml, 0.2 M
Tris, pH 8.5, 3–5 crystals of NaN 3 , ddH 2 O.
Preparation of Gelvatol: Add PVA to glycerol followed by
ddH 2 O. Add 3–5 crystals of NaN 3 . Stir with low heat for a few
hours or until reagents dissolved. Clarify the mixture by
centrifugation at 5,000 × g for 15 min. Aliquot and store at
4°C. See Note 4 for details on Gelvatol preparation.
12. 3 M NaOH.
13. 1% SDS in ddH 2 O.
14. Tissue-Tek plastic slide boxes and slide holders (Fisher).
15. Siliconized 24 × 60 mm coverslips.
To prepare, load coverslips onto 24-slot Tissue-Tek slide
holder. In a fume hood, dip twice into 3% Sigmacote (Sigma)
in chloroform (dip 2×), then dip twice into 100% EtOH. Air-
dry coverslips in hood. Prepare several hours in advance to
allow suf fi cient time for drying.
Carry out all procedures at room temperature unless otherwise
speci fi ed.
1. Prepare plasmid DNA using Qiagen miniprep kit or equiva-
lent. Digest 5–10 m g of template DNA to completion using
restriction enzyme of choice. Con fi rm completeness of diges-
tion using gel electrophoresis.
2. Following digestion, add 0.5 m g protease K to the enzyme
digestion buffer and incubate at 37°C for 15 min.
3. Increase volume to 200 m l with DEPC-treated TE.
4. Extract once with 200 m l TE-buffered phenol, and then once
with 200 m l chloroform.
5. Precipitate with 600 m l volumes of EtOH and 20 m l DEPC-
treated 3 M NaOAc.
6. Spin for 15 min at maximum speed to collect pellet, and then
wash twice in 200 m l 70% EtOH prepared with DEPC-treated
7. Air-dry the pellet and resuspend at 1 m g/ m l in TE.
1. Amplify the probe template using primers that include the pro-
moter sequence for the RNA polymerase used for probe gen-
eration. Primers commonly used for this include M13 forward
and reverse, and primers targeting the T7, T3, and Sp6 RNA
polymerase promoter sequences (see Note 6 ). Use approxi-
mately 0.1 ng of plasmid template, running 25–30 cycles of
ampli fi cation.
2. Con fi rm that a correctly sized band is ampli fi ed using agarose
3.1. Probe Preparation
( See Note 5 )
21915 High-Throughput RNA In Situ Hybridization in Mouse Retina
3. Purify ampli fi ed DNA using a Qiagen spin column, eluting
4. Following puri fi cation of template DNA, synthesize probe by
mixing the following components in the order indicated. Use
RNAse-free aerosol tips for all procedures: 2 m l 10× RNA poly-
merase buffer, 2 m l 10× DIG NTP mix, RNAse-free ddH 2 O
(to 17 m l fi nal), 1 m l RNAse inhibitor, and 1 m l RNA poly-
merase (T7, T3, or Sp6) to 19 m l fi nal volume. Generate a
master mix using these speci fi cations when screening multiple
probes. Finally, add 0.5–1 m g of template in l m l TE. This reac-
tion can be performed in RNAse-free Eppendorf tubes or in
96-well RNAse-free PCR plates.
5. Incubate for 60 min at 37°C.
6. Add 2 m l RNAse-free DNAse. Incubate for 15 min at 37°C to
degrade probe template.
7. Run denaturing gel with RNA size marker to check probe yield
8. If probe yield and integrity are satisfactory, add 2.5 m l 4 M
DEPC-treated LiCl and 75 m l 100% EtOH to precipitate.
Vortex at maximum speed for 5 s. If probe synthesis is per-
formed in 96-well plate format, transfer the product to RNAse-
free Eppendorf tubes prior to precipitation.
9. Store at −80°C for at least 2 h. Precipitate by centrifuging at
10. Wash twice in 200 m l 70% EtOH prepared with DEPC-treated
water. Air-dry the pellet and resuspend at 1 m g/ m l in TE. The
probe can be stored in EtOH inde fi nitely, or in TE for at least
2 years at −80°C.
1. Remove eyes and embed directly in O.C.T. compound (VWR)
in Peel-A-Way disposable plastic mold (Polysciences) and snap
freeze on dry ice. Store block at −80°C prior to sectioning.
2. Allow block to warm to cutting temperature for a minimum of
3. Cut 15–20 m m sections using a cryostat or freezing microtome
onto Superfrost Plus slides (VWR, 48311-703) (see Note 8 ).
4. Air-dry sections for at least 20 min. Slides can be dried for sev-
eral hours if necessary. Do not allow to dry overnight, how-
ever. Use dried slides immediately for in situ hybridization
analysis or store at −70°C in sealed slide box (can store for
1–2 years without appreciable loss of signal). If using stored
sections for analysis, allow them to equilibrate to room tem-
perature in a closed slide box.
( See Note 7 )
1. Remove cornea, lens, and sclera from dissected eyes to create
eyecup preparation. Fix tissue by overnight immersion at 4°C
in 4% PFA in 1× PBS.
2. Transfer to 30% sucrose in 1× PBS for 24 h at 4°C.
3. Mount in O.C.T. compound and section as described for
4. Air-dry for at least 20 min. Slides can be dried overnight
if needed. Use immediately or freeze at −20°C as described
1. Before beginning: Remove all RNAse contamination from slide
racks and chambers by rinsing with 0.3 M NaOH, rinsing with
MilliQ water, treating with RNAseZap or 1% SDS in MilliQ
water, and then rinsing once again with MilliQ water. Wipe
clean with Kimwipes (see Note 9 ). Use DEPC-treated solutions
for all treatments prior to hybridization (see Note 10 ).
2. Fix in fresh 4% PFA in 1× PBS for 10 min.
3. Wash 3× with PBS, 5 min each (see Note 11 ).
4. If tissue was fi xed in 4% PFA before embedding, treat with
2 m g Protease K in PBS for 10 min, followed by 2× 5-min
washes in PBS, a 5-min re fi x in 4% PFA/PBS (see Note 12 ),
and 2× 5-min PBS washes.
5. Incubate for 10 min in a mixture of 270 ml DEPC-treated
water/30 ml l M triethanolamine, pH 8.0/0.75 ml acetic anhy-
dride. Mix solution in an RNAse-free glass bottle and mix well
by shaking after addition of acetic anhydride (see Note 13 ).
6. Wash 3× 5 min with PBS.
MilliQ water is adequate for this and all subsequent steps.
1. Place slides in a chamber constructed from a 245 × 245 mm
BD Falcon* Square BioDish XL (Fisher, 02-667-21) square
Petri dish on a raised platform constructed from two 2 ml
polystyrene pipettes taped to the surface of the chamber with
waterproof tape. Alternatively, these can be bonded directly to
the surface of the dish using chloroform (see Note 14 ).
2. Place 500–1,000 m l of hybridization buffer on slide. Cover sec-
tions completely (see Note 15 ).
3. Leave in humidi fi ed chamber (keep moist with strips of gel
blot paper soaked in 5× SSC) for at least 90 min.
4. Prepare siliconized 24 × 60 mm coverslips several hours in
advance to allow suf fi cient time for drying.
5. Pour off prehybridization solution and blot off edges by direct
touching to bench paper. To reduce costs, prehybridization
solution can be saved, stored at –20°C, and reused 3–4 addi-
tional times. Add 75–100 m l hybridization solution containing
of Immersion Fixed
22115 High-Throughput RNA In Situ Hybridization in Mouse Retina
200–300 ng/ml DIG RNA which has been heated at 80°C for
5 min, vortexed for 5 s, and then snap-chilled on ice. Add probe
along bottom, long edge of the slide.
6. Clean coverslips using blown compressed air or manual tapping
if visible dust is present.
7. Coverslip slides by slowly lowering down a siliconized coverslip.
Place long edge, in contact with probe, down fi rst and lower the
rest slowly using fi ngers or a bent needle. Go slowly, to avoid
trapping of air bubbles. Once on the slide, raise and lower the
coverslip a couple of times to mix the probe with the prehybrid-
ization solution that remains on the slide (see Note 16 ).
8. Place slides horizontally in a humidi fi ed chamber (use 20 slide
capacity microslide boxes) (VWR). If possible, place slides with
different probes in separate boxes. However, if running many
probes in one experiment, place abundant probes at the bot-
tom. Make sure that long edge of slides is not in contact with
the back of the box, as this can promote capillary transfer of
the hybridization buffer away from the slide. Insert at least
four blank slides (pushed all the way to the back of the box) to
avoid this problem (see Note 17 ). Place a couple of Kimwipes
(VWR) soaked in 5–10 ml 50% formamide/5× SSC in the
bottom of the box to ensure that slides do not dry out.
9. Seal with waterproof tape (incubate at 65–72°C overnight)
(see Note 18).
1. Place slides in rack, submerged in 5× SSC to remove coverslips.
If coverslips are slow in falling off, the solution temperature
can be increased to 65–70°C. Carefully remove slides from
solution with forceps, grasping the frosted end. Coverslips
should fall off into the solution when slides are lifted up (see
2. Transfer slides with forceps into metal racks.
3. Incubate in 0.2× SSC at 65°C for 1 h in a water bath. After
30 min, jostle the slides a bit to remove bubbles that may have
accumulated on the slides. Slides can be washed longer if
needed, but not longer than 3 h total (sections will often fall
off the slide if heated longer) (see Note 20 ).
4. Wash with RNAse buffer for 5 min at 37°C.
5. Wash in RNAse buffer containing 10 m g/ml RNAse A for
30 min at 37°C (see Note 21 ).
6. Wash in RNAse buffer for 5 min at room temperature.
7. Wash 2× for 30 min in 0.2× SSC, 65°C.
8. Wash in 0.2× SSC for 5 min.
9. Wash in B1 for 5 min.
3.7. Washes, Antibody
Binding, and Signal
10. Place 1 ml buffer B2 on horizontal slides for 1 h (see Note 22 ).
11. Place 0.5 ml anti-DIG Ab (1:5,000 in buffer B2) on each slide.
Incubate in humidi fi ed chamber at 4°C overnight (see
Note 23 ).
12. Wash with buffer B1 3× for 5 min.
13. Wash with buffer B3 for 5 min, keeping slides horizontal in
humidi fi ed chamber (use the same prehyb chamber), puddle
on buffer B4. Keep in the dark (reaction is photosensitive) at
RT (cover chamber with foil). Check color after 15 min, 1 h,
and then again after 3 h and 6 h using a low-power microscope
(see Note 24 ). Can leave reaction for up to 3 days at either
room temperature or 4°C, and for even longer if background
1. Rinse slides in TE.
2. Rinse slides in ddH 2 O.
3. Mount in 4 drops Gelvatol per slide, using 24 × 60 mm cover-
slips. Leave overnight before examining. Once dry, wash away
excess Gelvatol with tap water, and then air-dry (see Note 25).
1. DEPC is highly toxic. Use caution when preparing solutions,
and do not breathe vapor. Autoclaving will fully inactivate 0.1%
DEPC, and the faint smell detectable after autoclaving re fl ects
residual ethanol contamination.
2. DEPC reacts with amine, hydroxyl, and thiol groups of pro-
teins, thus inactivating RNAse, as well as any other protein
with which it comes into contact. As a result, it is highly toxic
and should be handled with great care.
3. The fi lter unit used to prepare B3 can be wrapped with para fi lm
4. The viscosity of the Gelvatol requires optimization for each
batch prepared. When making the solution, add PVA in step 4
until the solution is clear and is slightly less viscous than molas-
ses. Then refrigerate the beaker of Gelvatol overnight at 4°C
(after step 5 and before step 6), and check it the next morning
to be sure that the viscosity is that of molasses. If it is, continue
on to step 6. If it is too viscous, add a little more glycerol to
lower the viscosity and then go on to step 6. If it is not viscous
enough, add more PVA with heat and refrigerate for a few more
hours, again checking the viscosity before going on to step 6.
Continue until viscosity is optimal. Gelvatol can be stored at
4°C inde fi nitely, or at room temperature for 1 month.
22315 High-Throughput RNA In Situ Hybridization in Mouse Retina
5. DNA templates used for cRNA probe synthesis should be
between 300 and 2,000 bp in length, with 700–1,000 bp being
optimal. Probe sequences should lack any repeat sequences
longer than 40 bp, and should not show more than 90% iden-
tity over any continuous stretch of 150 bp or more, or cross-
reactivity will result. Probe templates must be cloned into
vectors (such as pBluescript) in which the insert is fl anked by
T7, T3, or Sp6 RNA polymerase promoters, so as to allow
generation of labeled cRNAs. Many 3 ¢ directed ESTs, such as
those from the BMAP project or other large-scale cDNA
sequencing efforts, work very well as ISH probes, and can be
easily ordered from companies such as Thermo-Fisher.
Alternatively, PCR ampli fi cation followed by cloning into an
appropriate vector (such as the TOPO-TA vector) can be used
if repeat-free ESTs are not already available as probe templates.
Only cloned sequences should be used as templates for probe
generation. While PCR can be used to simultaneously amplify
template sequences from complex target preparations (e.g.,
reverse-transcribed cDNA), this introduces a strong possibility
of contamination. Once a clone containing the probe sequence
has been obtained, a linearized template must be generated in
order to conduct run-off transcription for cRNA synthesis. All
experiments should include both a positive and a negative con-
trol sample. Positive control probes should robustly recognize
target mRNAs in retina, but not be so abundant that contami-
nation of other slides is a real risk. Good negative control
probes should target transcripts that are not detectably
expressed in retina (such as albumin, cardiac albumin, GFP,
etc.). Use of sense control probes is not usually recommended,
as a sizeable minority of mammalian genes have associated
antisense transcripts ( 11 ) .
6. When using PCR primers that target an RNA polymerase pro-
moter sequence (i.e., T3, T7, or Sp6), it is important to include
2–3 bases of 5 ¢ overhang outside the primer sequence. This
substantially improves the ef fi ciency of cRNA synthesis.
7. Fixed tissue generally produces weaker signal intensity than
fresh-frozen, though use produces superior morphology and
better overall signal–noise ratio. Fresh-frozen tissue is often
easier to section consistently than fi xed tissue, and is particu-
larly useful for analyzing embryonic samples.
8. It is advantageous to fi ll as much as the slide as possible with
sectioned samples (although make sure that O.C.T. from other
sections does not overlap your tissue). The more sections pres-
ent on a slide, the greater the likelihood of obtaining high-
quality data in a given experiment. It is also the best way to
directly compare results from different samples, since placing
them on the same slide effectively eliminates slide-to-slide vari-
ation in signal intensity and probe spreading.
9. RNAses are very resistant to inactivation, so pretreatment is
designed to simply denature and remove any proteins that
might come into contact with the slides. In this step, this is
accomplished by treatment with detergent and a strong base.
10. Avoiding RNAse contamination during the fi rst day of the
procedure is critical. RNAses can come from human and ani-
mal skin and hair, or very often as contamination from other
experimental procedures, particularly plasmid DNA prepara-
tion. The danger is greater to the probe than to the sample, as
target RNA sequences in fi xed tissue are much less accessible to
RNAses than are cRNAs in solution. Do everything possible to
avoid these sources of contamination. Wear a clean lab coat
to cover arms, tie back long hair, and avoid working in lab
areas where animals are sacri fi ced. Use aerosol tips for all solu-
tions that require pipetting. Put down fresh bench paper before
beginning the procedure, and change gloves whenever the
possibility of contamination arises. We have often used a fume
hood to conduct all the steps up until the prehybridization
step, which both protects your sample from airborne debris
and removes any PFA vapors from the work area.
11. Dunk sections several times during each wash to ensure good
12. This re fi xation is important to maintain tissue integrity follow-
ing the protease K treatment.
13. Acetic anhydride modi fi es positively charged amine groups in
proteins and lipids, greatly reducing nonspeci fi c binding by the
negatively charged cRNA probe. However, acetic anhydride
hydrolyzes rapidly in aqueous solution, and it is extremely
important that the solution be applied immediately to the
slides once mixed.
14. It is important that slides rest above the surface of the box so as
to avoid capillary transfer of solution away from the sections.
15. Do not allow the tissue to dry out at this or any other succes-
sive stage. This will result in a loss of signal in the affected area.
Chill prehybridization solution on ice prior to applying to
reduce surface tension and ensure an even spread. Solution
used for prehybridization can be reused 3–4 times. Pour into
50 ml conical tubes prior to adding probe and store at −80°C
(reused solution should not be mixed with probe and used for
16. Probe must be well mixed to ensure even signal intensity across
17. It is critically important to change gloves between probes.
Failure to do so will result in cross-contamination.
18. 70–72°C is an optimal hybridization temperature for perfect
match probes that are longer than 300 bases. 65°C is better for
22515 High-Throughput RNA In Situ Hybridization in Mouse Retina
shorter probes or an imperfect match (i.e., rat probe used on
19. Do not use any force or pressure to remove the coverslip.
Doing so will result in damage to the tissue.
20. In this and every subsequent step, preheat solutions in a water
bath prior to washing.
21. The RNAse treatment degrades the single-stranded cRNA
probe while leaving double-stranded RNA–RNA hybrids
intact. This results in a considerable reduction in background
signal. However, it requires great caution, as the RNAse used
in this step can contaminate materials used prior to the hybrid-
ization step. Every effort should be made to prevent this. Make
sure that slide boxes and racks used for RNAse treatment are
treated with 0.3 M NaOH and RNAseZap after use. Change
gloves after handling slides during this step.
22. To ensure even spread of solution across the slide and to reduce
the amount of serum used, chill B2 solution on ice prior to
23. For abundant RNA a 1-h incubation is suf fi cient, but over-
night incubation greatly enhances the signal and reduces color
24. Optimizing the ratio of target to background signal is crucial
for obtaining high-quality data using this procedure. To this
end, it is essential to vigilantly track the progress of the color
reaction so as to assess when it should be terminated. The most
ef fi cient way to do this is to directly view the slides in their
chamber against a white background using a dissecting micro-
scope such as a Leica Stemi 2000-C or an Olympus BH-2 at
roughly 10× magni fi cation. Stop the reaction before any obvi-
ous background is visible, but slightly past the point at which
the color exposure appears optimal to the eye, as the cellular
signal intensity will look weaker at the 200× magni fi cation usu-
ally used for photographing retinal sections.
25. Gelvatol automatically seals the slide once it dries, which
reduces labor substantially when running large number of
slides. One can also use Aquamount (Fisher, 14-390-5) or an
equivalent water-based solution to mount the slides, but they
must then be sealed with clear nail polish to prevent drying
during long-term storage.
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