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In Situ Hybridization with Oligoprobes Targeting the 16S rRNA
1. Make 2.0N NaOH (100mL):
a. Dissolve 8g NaOH in 100mL milliQ-water
b. Keep at room temperature in a plastic container
c. A glass container might result in formation of precipitates.
2. Make 3X PBS, pH 7.2 (500mL):
a. This solution can be bought or made by you.
b. The concentration should be:
i. 390mM NaCl
ii. 21mM Na2HPO4
iii. 9mM NaH2PO4
c. Weigh out:
i. 11.39g NaCl
ii. 1.491g Na2HPO4 • 7H2O
iii. 0.621g NaH2PO4 • H2O
d. Dissolve all these in 450 mL milliQ-water
e. With NaOH 2.0N, adjust pH at 7.2
f. Complete volume to 500mL
g. Filter with 0.22 microns unit filter
h. Store at 4oC
i. Used within a month
3. Make 4% PFA (paraformaldehyde) / PBS pH 7.2 (25mL):
a. Dissolve 1 g PFA in 15mL milliQ-water at 60oC
b. Add 1 drop NaOH 2.0N
c. When the solution is perfectly clear let it cool down at room temperature.
d. Add 10mL of cold 3X PBS pH 7.2
e. Check pH and adjust to 7.3 with HCl 0.01N
f. Final volume should be 25mL
g. Filter the solution with a 0.22 microns filter plugged to a syringe
h. Use within 24 hours after preparation to minimize autofluorescence.
i. For cold fixarion method keep solution on wet ice before use.
i. Incubate sample at 4oC for 1 hour per millimeter of sample
ii. The cold fixation helps with keeping the structure intact.
iii. Or Incubate sample at 50oC for 20 minutes.
iv. If you use standard method for sample fixation (4oC for 3hrs) keep
solution on wet ice before use.
4. Make deionized formamide:
a. For hybridization steps, it is strongly recommended to use deionized
formamide.
b. Some ionic species in formamide interfere with the hybridization step and
can be removed by purifying it with a resin.
c. The most reliable resin is: The molecular biology grade AG501-X8 and
Bio-Rex MSZ 501 (D) mixed bed resin (20-50 mesh) from Bio-Rad.
d. It consists of equivalent amount of AG50W-X8 strong cation exchange
resin H+ form and AG1-X8 strong anion exchange resin OH- form.
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6.
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e. In a glass beaker mix 50g resin with 1 Liter of formamide.
f. With the stirrer agitate at least for 1 hour at room temperature (under the
chemical hood).
g. Filter the deionized formamide
h. Store in Falcon tubes at -20oC.
Prepare granules:
a. Should be done relatively fast because rRNA is the target and will be
degraded with time if granules are left dying.
b. Granules should be handled with great care, especially to avoid
degranulation and/or damaging of the surface area.
c. Don’t need to use RNAase free treated buffers
d. Always work with clean and autoclaved/filtered (whenever possible)
materials and solutions until fixation step is over.
e. Fresh granules are easier to work with and give better results compared to
frozen granules.
f. If you need to freeze granules, biofilms or bacterial cultures, quick
freezing with liquid nitrogen to prevent ice crystals.
Ways to freeze your samples for in situ hybridization:
a. There are many ways to freeze samples
i. Ethanol/dry ice (-80o)
ii. Dry ice (-80oC)
iii. Chilled isopentane (by liquid nitrogen) (-163oC)
iv. Liquid nitrogen (-196oC).
b. Freezing methods give superior morphology with increasing rapid freezing.
Sample fixation with 4% PFA (paraformaldehyde) / PBS pH 7.2:
a. Washed Granules in ice-cold 1X PBS pH 7.2
b. Let the granules settle down for only 5 minutes in the buffer
c. Rinse off the solution with a pasture pipette.
d. Repeat for steps A-C 2 or 3 times more
e. Incubate the cleaned granules in fresh cold 4%PFA (paraformaldehyde) /
PBS pH 7.2.
f. Penetration of fixative into sample is 1mm/hour at 4oC.
g. Longer time in fixation may damage the structure and cause more
autofluorescence from degradation products of the non-stable
paraformaldehyde.
h. Rinse the granules to remove any fixative.
i. Let the granules settle in ice-cold 1xPBS pH 7.2 for 5 minutes
j. Rinse off the solution with a Pasteur pipette.
Embedding with OCT for cryosectionning:
a. Cryostat: is an apparatus working at freezing temperatures and it capable
of slicing samples and thin as 2microns.
b. When you use the cryotome, it is important to vary the temperature and
not reply on an untested preconception of the ideal temperature.
c. For thinner sections it is better to use lower temperatures.
d. At the same low temperature a thicker section may tend to fragment or
crack.
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e. Before slicing the frozen sample with the cryostat:
i. Leave the block to be sliced in the cryotome at -20/-25oC for at
least 30 minutes to make sure that the sample won’t be at -80oC
otherwise it will crack.
ii. Make sure that the blade and anti-roll plaque are clean.
1. Use acetone and a Q-tip to clean it before, during and after
the slicing so there won’t be any fat residue on them.
iii. Read the troubleshooting section at the end of the cryostat user’s
manual (this will save you frustrations).
iv. Treat with a cryoprotectant:
1. Quick treatment:
a. Let settle down fixed and rinsed granules in cold
solution of 4oC filtered 20% sucrose/1x PBS pH 7.2
b. As soon as the granules have sunk, they can be
drained on a filter paper
c. Place directly in a mold containing some tissue-tek
OCT compound.
d. Placed the mold in chilled isopentane followed by
liquid nitrogen or directly in liquid nitrogen.
2. Long treatment:
a. Mix sucrose with OCT
b. Add granules
c. Embed and cryoprotect at room temperature
overnight for each step.
i. Day 1 - 100% of 15% sucrose
ii. Day 2 - 75% of 15% sucrose and 25% of
pure OCT
iii. Day 3 - 75% of pure OCT and 25% of 15%
sucrose
iv. Day 4 - 100% of pure OCT
v. Day 5 - freeze the samples in liquid nitrogen.
f. Best slicing occurs from 5 to 10 microns.
g. A five micron section is acceptable for further epifluorescence of CLSM
analysis of samples.
h. Never leave any sample in the cryostat because it is programmed to
defrost at midnight and you will lose your sample.
9. After the slices are placed on the Poly-L-lysine slides, there are 2 choices:
a. Air dry the slides 4 to 6 hours before dehydration step.
b. Keep the slides in a dessicator box in a freezer at -80oC until ready for
dehydration.
i. If you choose another method, just make sure there won’t be any
ice crystal formation during the storage.
10. Preparing samples to hybridize:
a. If planning for shipping before 2 o’clock the next day, leave in frozen
deionized formamide at 4oC the night before
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i. It will be ready to warm at 46oC and add to the hybridization mix
the next morning.
b. At this point the selected granules on sections should have been:
i. Observed with a microscope equipped with a graticule
ii. Measured
iii. Described with a drawing.
iv. Locations of granules on each section and on each numbered
microscope slide should be clearly identified in the lab book.
1. Always use a pencil to identify the frosted part of the slide
2. Even indelebile marker will get erased by xylene and
ethanol prior hybridization step.
c. After removing paraffin precisely circle (under the slide) with a fine tip
indelebile marker each granule to be hybridized
i. The experiment will take place under minimal light and granules
are very hard to see.
d. Just prior covering granule with probes, circle the granule area with an
immedge / hydrophobic barrier pen so that cross contamination of probes
between granules won’t occur.
i. That is especially helpful if more than 20 uL is used per sample.
11. Make hybridization solution for fluorescent probes:
12. Hybridize granules with fluorescent probes:
a. Each granule is covered with 10 microliters of hybridization mixture with
probe.
b. Hybridization step is 2 hours at 46oC in a humid chamber (Tupperware)
i. Some mix without probe will be poured on a piece of labcover at
the very bottom of the humid chamber (Tupperware).
13. Make washing solution for fluorescent probes:
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14. Washing for 20 minutes at 48oC in humid chamber.
15. Dry the slides under chemical hood and in the dark.
16. Rinse the granule with some drops of equilibrium buffer provided by molecular
probes.
a. Milli-Q water at room temperature or colder
17. Add 1 drop of antifade reagent in glycerol
18. Gently put a coverslip on.
19. Use nail polish to seal the edge of the coverslip.
20. Make sure it is dry before examination under the microscope.
21. If you need to ship the samples:
a. Place the box containing the slides between 2 icepaks (freeze the night
before) in a Styrofoam container.
b. Fill out the forms and write down instructions for handling and storage.
c. Place also in the container a sheet in plastic bag describing exactly what is
sent.