In Situ Hybridization Protocol (for plant roots)

This protocol is a modification of several protocols: 1. Jackson (1991). In situ hybridization in plants. In Molecular Plant Pathology : A Practical Approach (Oxford University Press), pp. 163-174
2. Lincoln et al. (1994). Plant Cell 6, 1859-1876.
3. Coen et al. (1990). Cell 63, 1311-1322

note: All solutions, glassware, forceps, etc. must be RNase free up to hybridization step.

I. Fixation, Embedding, and Sectioning
1. Put Fix solution in a glass vial
2. Put tissue in Fix Solution and vacuum (~760 Hg mm Vac.) for 15~20 min.
3. Swirl the solution and check if the tissue sinks to the bottom (if not, repeat the vacuum step)
4. Replace solution with new Fix Solution and store overnight at 4°C
5. Put tissue in 1X PBS for 30 min.
6. Repeat step 5
7. Embed tissue in 1% Agarose and trim into a block (fig. 1) -Alternatively, this step can be done after the 70% ethanol step 8. Put tissue block through an ethanol series (30 min. each) :
1-30% 2-50% 3-70% 4-85% 5-95% 6-100% 7-100% 8-100% 9 1/2 100 1/2 Hemo-De 10-Hemo-De 11-Hemo-De 12-Hemo-De

9. Put tissue block in 1/3 Hemo-De and 2/3 solid Paraplast chips (Xtra) and store at room temp. overnight
10. Put tissue block in 60°C for ~2 hrs.
11. Replace solution with freshly melted Paraplast for 2 hrs. or longer at 60°C
12. Repeat step 11 at least three times
13. Transfer the tissue block to a Peel-A-Way mold and fill the mold with melted Paraplast to the top
14. Store at 60°C for 2hrs. or longer
15. Orient the tissue block in the mold and put on ice for ~1 hr.
16. Section (thickness should be ~10mm)
17. Put the sectioned ribbon on preheated water (42°C) for ~1 min. or wait until the ribbon is wrinkle-free
18. Rescue ribbon on a slide(Superfrost) and tap the slide on it’s side to release air bubbles and liquid trapped underneath the ribbon
19. Put slide on a slide warmer (42°C) overnight
20. Label and store slides in a dry slide box

II. Probe Synthesis

1. Linearize template for making both anti-sense and sense probes
Note: only use enzymes that create 5′ overhang
2. Check digests on a gel
3. Phenol/Chloroform and precipitate
4. Resuspend in depc-water
5. Setup for transcription
2 m1 mg/ml BSA
2 ml 10X Transcription Buffer
2 ml 10% Triton X-100
2 ml 10X dig RNA labeling mix
1 ml RNase Inhibitor
up to 9 ml of template ( total of 1 mg)
2 ml RNA Polymerase (T7, T3, SP6)
incubate for 2 hrs. at 37°C
6. DNase Treatment
1 ml RNase Inhibitor
5 ml 10X DNase Buffer
2 ml depc-water
1 ml DNase I, RNase Free (23u)
incubate for 15 min. at 37°C
7. Stop the reaction and precipitate
2 ml 0.5M EDTA
6 ml 4M LiCl
180ml chilled 100% EtOH
incubate overnight at -80°C
8. Spin for 15 min.
9. Discard solution and put 200 ml of chilled 70% EtOH
10. Spin for 5 min.
11. Dry and resuspend pellet in 95 ml depc-water
12. Add 1 ml RNase Inhibitor and incubate for 30 min. at 37°C
13. Run 10 ml on a RNA gel to check the transcript
14. Quantify probe according to the Genius System User’s Guide
15. Use 20 ng~70 ng of probe/ slide

III. Pretreatment

1. Put the slides in a slide rack
2. Hemo-De 10 min.
3. Hemo-De 10 min.
4. MeOH 15 min.
5. 100% EtOH 1 min.
6. 100% EtOH 1 min.
7. 95% EtOH 1 min.
8. 85% EtOH in .85% NaCl 1 min.
9. 70% EtOH in .85% NaCl 1 min.
10. 50% EtOH in .85% NaCl 1 min.
11. 30% EtOH in .85% NaCl 1 min.
12. 0.85% NaCl 2 min.
13. 1X PBS 2 min.
14. 0.2M HCl 20 min.
15. H2O rinse
16. 2X SSC 20 min.
17. H2O rinse
18. 10mg/ml Proteinase K 30 min. at 37°C in 0.1M Tris-Cl(pH 7.5)/ 0.05M EDTA(pH 8.0) note: preheat solution to 37°C before adding Proteinase K
19. 1X PBS 2 min.
20. Fix Solution 10 min.
21. 0.5% Acetic Anhydride 10 min.
in 0.1M Triethanolamine( pH8.0)
note: add Acetic Anhydride one drop at a time while spinning the solution with a stir bar
22. 1X PBS 2 min.
23. 0.85% NaCl 2 min.
24. Dehydration: repeat steps 5 to 11 backwards
note: can reuse EtOH solutions except 100% EtOH
25. Store overnight at 4°C in a container with 100% EtOH covering the bottom or go on to the next step

IV. Pre-Hyb. and Hybridization

1. Make Pre-hyb solution and setup a container for the slides (fig. 2)
2. Put the slide in the container
3. Apply 200ml/slide of pre-hyb solution and cover with Parafilm
note: Be gentle when covering with Parafilm to avoid air bubbles
4. Store slide in the container at room temp. for 1 hr.
5. Prepare hyb solution
6. Move container to 50°C for 1 hr. or longer
7. Put hyb solution at 65°C for 10 min. and keep on ice
8. Remove Parafilm and blot the edge of the slide on a paper towel to allow the pre-hyb solution to drain
9. Put the slide back into the container
10. Apply 200ml/slide of hyb solution to each slide and cover with parafilm
note: setup is exactly like the pre-hyb step
11. Store slides in the container at 50°C overnight

V. Washing

1. Remove Parafilm and put the slides back to the slide rack
2. Rinse in 5X SSC/ 50% Formamide
3. Replace solution with new 5X SSC/ 50% Formamide and store at 50°C for 4~5 hrs.
4. Rinse in NTE solution 1~2 times
5. Place slide in 10 mg/ml RNase A in NTE at 37°C for 30 min.
note: Preheat NTE solution to 37°C before adding RNase A(same as Proteinase K step)
6. Wash in NTE at 37°C for 5 min.
7. Repeat step 6 twice
8. Wash in 0.5X SSC / 50% Formamide at 50°C for 1 hr. or longer
9. Rinse in 1X PBS for 5 min.
10. Store overnight at 4°C in 1X PBS or go on to the next step

VI. Detection

1. Make Blocking Solution 2. Incubate in Blocking Solution for 45 min. on a rocker/shaker
3. Make Buffer A
4. Incubate in Buffer A for 45 min. on a rocker/shaker
5. Setup a container for step 6
note: setup is same as the pre-hyb and hyb step but instead of using Formamide, use Buffer A to wet the bottom
6.Put the slide into the container
7. Apply 500ml/slide of dilute antibody-conjugate(1ml antibody/ 1ml Buffer A) and cover with Parafilm
8. Incubate slide in container at room temp for 1 hr.
9. Discard the Parafilm and put the slide back on the slide rack
10. Wash in Buffer A for 20 min. on a rocker/shaker
11. Repeat step 10 three times
12. Wash in Detection solution for 5 min.
13. Repeat step 12
14. Put the slide back into the same container made at step 5
15. Apply 500ml detection buffer/ slide and cover with Parafilm
16. Incubate for ~2 days in the dark
17. Remove Parafilm and rinse slide in TE
18. Drain excess liquid and apply 3 drops of Aqua-Polymount/ slide
19. Put glass coverslip

Solutions

note: All solutions must be RNase free up to hybridization step
1. Fix Solution
1. Add 4g Paraformaldehyde to 50 ml H20
2. Stir at on a hot plate and wait for the solution to come to 65°C
note: Work should be done in a fume hood Paraformaldehyde vapor is toxic
3. Add 6 ml 10M NaOH and stir until the solution becomes clear
4. Filter through a Whatman filter paper
5. Add 10 ml 10X PBS
6. Add 40 ml H20
7. Store at 4°C for up to 1 day
2. 10X PBS (1L)
76g NaCl
18.8g Na2HPO4�7H20
4.1g NaH2PO4�H20
adjust to pH 7.0

3. Pre-hyb Solution ( for 20 slides)
Formamide 2 ml
10X Salts 400 ml
50X Denhardt’s Solution 80 ml
tRNA (20mg/ml) 40 ml
RNase Inhibitor 2 ml
dH20 1480 ml

4. Hyb Solution (for 1 slide)
Hyb Mix 160ml
Formamide 20 ml
Probe 20 ml(x ml probe/ up to 20 ml dH20)

5.Hyb Mix (for 20 slides)
Formamide 2 ml
10X Salts 500 ml
50% Dextran Sulfate 1 ml
note: takes long time to go into solution prepare in advance
poly A (10 mg/ ml) 250 ml
tRNA 50 ml
50X Denhardt’s Solution 100 ml
RNA inhibitor 2.4 ml
dH20 97.6 ml

6. 10X Salts (10 ml)
1.75g NaCl
0.121g Tris
0.12g Sodium Phosphate
1 ml 0.5 M EDTA

7. 50X Denhardt’s (50 ml)
0.5g Ficoll (type 400,Pharmacia)
0.5g Polyvinylpyrrolidone
0.5g BSA (Fraction V, Sigma)

8. NTE (1L)
100 ml 5M NaCl
10 ml 1M Tris pH 8.0
10 ml 0.5M EDTA

9. Blocking Solution (200 ml)
1g Blocking Reagent
20 ml 1M Tris pH 7.5
6 ml 5M NaCl
600ml Triton X-100

10. Buffer A (1 L)
10g BSA
3 ml Triton X-100
100 ml 1M Tris pH 7.5
30 ml 5M NaCl

11. Detection Solution (1 L )
100 ml 1M Tris pH7.5
20 ml 5M NaCl
pH to 9.5
50 ml 1M MgCl2

12. Detection Buffer (10 ml)
9.7 ml Detection Solution
200 ml NBT/BCIP Solution
100 ml 24mg/ml levamisole

13. 10X DNase Buffer (100 ml)
40 ml 1M Tris pH 7.5
6 ml MgCl2
pH to 7.5