IN SITU Sagittal Section 1 of 5
Sagittal Section IN SITU
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All glassware must be baked O/N @ 180˚C for min. 8 hours.
Orange caps/plastic rings should be treated with RNase away, rinsed with distilled
water and autoclaved. Don’t forget stir-bars!
Gloves MUST be worn at all times.
Solution:
 0.1% (v/v) DEPC-treated water:
Fill 1L bottle with milli-Q water up to the mark. Add 1 mL DEPC (stored in
fridge). Add a stir bar. Let stir O/N. Autoclave the next day. Do not remove the
stirbar.
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In Situ Hybridization (ISH) fixative:
Make a solution containing 85% EtOH, 10% formaldehyde, and 5% glacial acetic
acid.
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EtOH’s (95%-70%-50%) make right before use.
Prepare in DEPC-treated water. These are used to rehydrate dewaxed specimens
and can be reused several times.
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10X PBS
Dissovle 20 g sodium chloride, 0.5 g potassium chloride, 2.88 g sodium
phosphate dibasic, 0.5 g potassium phosphate monobasic in DEPC-treated water.
Filled up to 250 mL. Autoclaved!
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4% Paraformaldehyde (PFA)
Use special glassware set aside for PFA. Do not use for anything else as it is
useless. Can be prepared the day before use and stored O/N @ 4˚C. Dissolve 10
g PFA in 100 mL DEPC-treated water at 55-60˚C with stirring. Add 10M NaOH
until solution clears. Remove from heat and add 90 mL DEPC-treated water and
10X PBS. Cool to room temperature and filter thru #1 Watman paper.
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1M Tris (pH 7.5)
Dissolve 60.6g Tris base in 400 mL DEPC-treated water. Add 32.5 mL
concentrated HCL and fill up to 500 mL. Check pH with test stripe. Autoclave!
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1M Tris (pH 8.0)
Dissolve 60.6g Tris base in 400 mL DEPC-treated water. Add 21 ml
concentrated HCL and fill up to 500 mL. Check pH with test stripe. Autoclave!
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TBS
Add 50 mL of 1M Tris (pH 7.5) and 30 mL SM NaCL, filling with DEPC-treated
water up to the 1L mark.
200 mM HCL
Add 4 mL []ed HCL to 246 mL water.
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IN SITU Sagittal Section 2 of 5
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Acetic anhydride (make right before use)
Add 25 mL of 1M Tris (pH 8.0), 224 mL water and 1.25 mL acetic anhydride.
This helps to block positive charges that might increase background.
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Proteinase K
Measure 125 μl of 20 mg/mL Proteinase K stock. 2.5 mL of 1M Tris (pH 8.0),
0.5 mL of 0.5M EDTA and fill with DEPC-treated water up to 250 mL.
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Hyb Buffer
Measure a volume of 2.5 mL formamide, 10 μL 10% SDS, 50 μL 10 mg/mL yeast
tRNA, 1 mL 50% dextran sulfate, 0.5 mL 20X SSC, and 985 μl (or 0.985 mL) of
DEPC-treated water.
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Probe preparation
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0.5 M EDTA (pH 8.0)
Add 18.61 g EDTA to 80 mL DEPC-treated water. Stir vigorously and add 2 g
NaOH pellets. Check pH and fill to 100 mL. Autoclave!
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5 M NaCl
Dissolve 73.1 g in 200 mL DEPC-treated water. Fill to 250 mL and autoclave!
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20X SSC
Measure 87.7 g sodium choride and 44.1 g sodium citrate dihydrate in 400 mL
DEPC-treated water. Adjust pH to 7.0 with 10 M NaOH. Fill to 500 mL.
Autoclave!
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Humidifier chambers
3 petridish plates. Cover bottom with Watman filter paper. Saturate it generously
with DEPC-treated water. Using 2 strips of thin tubing to make a platform to lay
the slides on.
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Roche blocking agent
Add 1 mL of the Roche block stock, 1 mL of Fetal Calf Serum (FCS), 100 μl
sheep serum and 7.9 ml maleic acid.
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Anti-DIG 1:1000 in blocking agent
Add 1 mL of Roche blocking agent, 1 mL of Fetal Calf Serum (FCS), 8 ml of
maleic acid and 10 μL of antibody DIG.
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Maleic Acid Buffer
Add 3.45 g maleic acid, 2.19 g NaCl, and fill up to 250 mL with DEPC-treated
water. Autoclave!
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IN SITU Sagittal Section 3 of 5
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AP Buffer
Add 3.03 mL of Tris (pH 9.5), 2.54 g MgCl2, 1.46 g NaCl and fill up to 250 ml
with DEPC-treated water. Autoclave!
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Tris (pH 9.5)
Measure 3.03 g Tris Base, add HCL for pH and fill up to 25 ml with DEPCtreated water. Autoclave!
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IN SITU Sagittal Section 4 of 5
Day 1:
 Use GLOVES at all times.
 Use sections that were prepared in ISH fixative.
 All solutions are made in DEPC-treated water.
 Color frost +/plus slides were used.
 Each container holds about 250 mL solution. Perform Day 1 under the hood in Dr.
Scemama’s Lab.
1. Clean all surface areas and container with RNase-away.
2. Dewax the slides in Xylene 2X – 15 minutes each ( use around 8 slides)
3. Perform the following rehydration rinses @ RT:
a. 100 % EtOH
2 minutes
b. 95% EtOH
2 minutes
c. 70% EtOH
2 minutes
d. 50 % EtOH
2 minutes
e. DEPC-treated water
2 minutes
f. 1X PBS *
2 minutes
* 1X PBS made by adding 25 mL of 10X PBS stock and filling it up to 250
mL with DEPC-treated water.
4. Post-fix:
a. 1X – 4% PFA
20 minutes @ RT (set aside bottle just for PFA)
IMPORTANT: Save the 4% PFA for Day 3
Turn on slide moat @ 55˚C and add DEPC-treated water in every hole
using plastic pipette to HUMIDIFY!
5. Wash:
a. 3X TBS
5, 10, 10* min @ RT
b. 1X 200 mM HCL
10 min @ RT (neutralize w/ NaOH until pH 6-8 and dump down sink)
c. 3X TBS
5, 10, 10 min @ RT
* Meanwhile, prepare Acetic Anhydride. Put in a stirbar.
6. Wash:
a. 1X Acetic Anhydride
10 min @ RT (w/ vigorous stirring)
a. 3X TBS
5, 10, 10 min @ RT. Put TBS in 4˚C.
a. 10 μg/mL Proteinase K
15 min @ RT. (Preheat heat block @ 95˚C for step 13)
7. Wash:
8. Add:
9. Wash:
a. 3X - 4˚C TBS
5, 10,10* min @ RT
* IMPORTANT
10. Important: prepare probes! Make enough for an extra slide.
11. Using Kimwipes, remove most of the TBS. Make sure slide moat is on!
12. Using pipette, add probe evenly of the slides (about ~ 60 μl).
13. Place coverslip over the slides. Put on heat block for 4 minutes @ 95˚C.
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IN SITU Sagittal Section 5 of 5
14. Once, 4 minutes are up, remove slides from the heat block and seal slide with rubber
cement using a syringe (no needle needed).
15. Make sure the slide moat is on and set at 55˚C. Also, make sure water was added into
each of the wells in order to humidify.
16. Set the slides on the humidifier for O/N @ 55˚C.
Day 2:
 Thaw out the Formamide. Prepare 1:1 formamide: 2X SSC and stick in @ 55˚C.
 Will need a coaklin jar (holds about 50 ml solution).
 Clean everything w/ RNase away. Wear GLOVES!
1. Using forceps/tweezers, carefully remove the rubber cement from all around the
slides.
2. Remove coverslip which will slide off easily.
3. Put the slides in 2X SSC (in coaklin jar) immediately after removing the coverslips.
Make sure slides w/ different probes face in opposite direction. Prepare Roche
blocking agent for step 8.
4. Wash (w/ agitation):
a. 3X
20 mins
“1:1 Formamide: 2X SSC” @ 55˚C
b. 2X
15 mins
1X SSC @ RT (leave door slightly open & turn temp. of incubator to its lowest.)
c. 3X
5 mins
TBS @ RT
5. Using Kimwipes, gently wipe off excess TBS off the slides.
6. Create humidifier chamber
7. Using a PAP pen, circle the entire region of section. This will act like a hydrophobic
barrier and keep blocking agent from flowing out.
8. Add enough of the Roche blocking agent, covering entire region within PAP pen
circle. Block for 1 hour in the humidifier chambers (petridishes) at RT.
9. Using vacuum filter, suction out all liquid off from slides using a pipette tip attached
to the end of vacuum filter.
10. Meanwhile, 15 minutes prior to stop time, prepare the anti-DIG 1:1000 in blocking
agent.
11. Add enough of the anti-DIG 1:1000 to cover the slide within PAP pen circle, and
incubate for 1 hour @ RT.
12. REPEAT step 9.
13. Wash:
a. 4X
TBS 5, 10, 10, 10 minutes
14. Equilibrate section in AP buffer for 2 min.
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