3.4 Pre-hybridization
Sample Preparation
1. Set up an incubator at 37
C. Pre-warm the TE-protease solution to 37
C.
2. Lay slides in a slide holder. Immerse the slides twice in HistoClear solution, 10 min each time.
3. Wash off the Histo-Clear solution by immersing the slides two
times in 100% ethanol for 1 min each time.
4. Rehydrate the slides by immersing them successively for 30 s in
each solution of 95%, 80%, 70%, 30%, and 10% ethanol
(vol/vol).
5. Rinse the slides with water for 1 min.
6. Wash the slides two times for 2 min with PBS.
7. Transfer the slides to the TE-protease buffer prewarmed to
37
C and incubate for 20 min (see Note 3).
8. Neutralize any formaldehyde autofluorescence by incubating
the slides in glycine solution for at least 2 min. This step can be
extended to 30 min.
9. Rinse the slides two times for 2 min with PBS (1Â).
10. Prepare fresh TAE solution during the previous step. Incubate
the slides in TAE solution for 10 min with gentle agitation (see
Note 4).
11. Wash the slides two times for 2 min in PBS (1Â).
12. Wash the slides in water for 1 min.
13. Dehydrate the slides by immersing them successively for 1 min
in each solution of 10%, 30%, 50% 70%, 80%, and 95% ethanol.
14. Incubate the slides two times for 1 min in 100% ethanol.
15. Store the slides in 100% ethanol for at least 2 h for sample
permeabilization. Slides may be stored at 4
C for up to
4 weeks.
3.5 Hybridization
1. Dilute the hybridization probe (see Note 5) to a stock concentration of 100 ng/μL using nuclease-free water. The starting
probe concentration should be 1 ng/μL for most plant tissues
(see Note 6). To prepare a hybridization mix, add 1 μL probe
stock to 99 μL hybridization buffer to make a working stock.
Mix the solution well by pipetting up and down several times,
without causing bubbles.
2. Take out the slides from the 4
C storage and warm up to room
temperature.
3. Wash the slides in washing buffer for 2 min.
4. Apply 100 μL hybridization mix to each slide.
5. Apply the membrane hybridization cover, and tap on the membrane as it is lowered down to completely wet the tissue. Try to
avoid bubbles.
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Kun Huang et al.
Sample Preparation
1. Set up an incubator at 37
C. Pre-warm the TE-protease solution to 37
C.
2. Lay slides in a slide holder. Immerse the slides twice in HistoClear solution, 10 min each time.
3. Wash off the Histo-Clear solution by immersing the slides two
times in 100% ethanol for 1 min each time.
4. Rehydrate the slides by immersing them successively for 30 s in
each solution of 95%, 80%, 70%, 30%, and 10% ethanol
(vol/vol).
5. Rinse the slides with water for 1 min.
6. Wash the slides two times for 2 min with PBS.
7. Transfer the slides to the TE-protease buffer prewarmed to
37
C and incubate for 20 min (see Note 3).
8. Neutralize any formaldehyde autofluorescence by incubating
the slides in glycine solution for at least 2 min. This step can be
extended to 30 min.
9. Rinse the slides two times for 2 min with PBS (1Â).
10. Prepare fresh TAE solution during the previous step. Incubate
the slides in TAE solution for 10 min with gentle agitation (see
Note 4).
11. Wash the slides two times for 2 min in PBS (1Â).
12. Wash the slides in water for 1 min.
13. Dehydrate the slides by immersing them successively for 1 min
in each solution of 10%, 30%, 50% 70%, 80%, and 95% ethanol.
14. Incubate the slides two times for 1 min in 100% ethanol.
15. Store the slides in 100% ethanol for at least 2 h for sample
permeabilization. Slides may be stored at 4
C for up to
4 weeks.
3.5 Hybridization
1. Dilute the hybridization probe (see Note 5) to a stock concentration of 100 ng/μL using nuclease-free water. The starting
probe concentration should be 1 ng/μL for most plant tissues
(see Note 6). To prepare a hybridization mix, add 1 μL probe
stock to 99 μL hybridization buffer to make a working stock.
Mix the solution well by pipetting up and down several times,
without causing bubbles.
2. Take out the slides from the 4
C storage and warm up to room
temperature.
3. Wash the slides in washing buffer for 2 min.
4. Apply 100 μL hybridization mix to each slide.
5. Apply the membrane hybridization cover, and tap on the membrane as it is lowered down to completely wet the tissue. Try to
avoid bubbles.
28
Kun Huang et al.