In Situ Hybridization: a Practical Protocol Using Biotinylated RNA Probes on Paraffin-Embedded

In Situ Hybridization: a Practical Protocol Using Biotinylated RNA Probes on Paraffin-Embedded

In situ hybridization: A practical protocol using biotinylated RNA probes on paraffin-embedded sections.

The following protocols were pre-tested for rat brain sections of 5m thickness and should be carefully checked before application to other tissues and probes.
4.1 Paraffin-embedded sections
(1)Dissect out whole brain into ice-cold phosphate buffered saline (PBS).
(2)Place tissues in an appropriate tube with 3.7% formaldehyde in PBS and leave

at room temperature 24-48 h.

(3)Successively replace solution with the following, each for at least 30

min and with occasional agitation: 70% ethanol (twice), 85% ethanol, 95%

ethanol, absolute ethanol (twice). Tissues can be stored indefinitely in any of the

alcohol solutions.
(4)Replace the solution with xylene, twice for 30 min each, then a 1:1

xylene: wax mix at 50 C for 20 min, followed by three changes of wax, each

for 20 min. It is important to not exceed these times, unless this is necessary for

larger tissues. Many alternative paraffin waxes can be used; we use pastillated

Fibrowax (BDH).
(5)Transfer the brain into a mold, orient and allow to set.

The tissue can be oriented with a warmed needle.
(6)Store at 4 C until required for sectioning.
(7)Cut ribbons of 5 m sections.
(8)Float sections on a bath of distilled water at 50 C until creases disappear, and

collect on submerged slides (see 3.2.).
(9)Dry slides at 37 C overnight and store dessicated at 4 C.

4.2. Slides used for in situ hybridization

In order to prevent the tissue from falling off the slides during in situ hybridisation and washings you should either pre-treat the slides with silane or use Super-Frost/Plus slides (Menzel-Glaeser, Germany).

Silanisation of slides

1.Microscope slides

Pre-cleaned Corning No. 9248, single frosted

2.Detergent treatment

10% Extran MAO1 cleanser (Merck, Darmstadt, Germany , cat. No. 7555), pre-warmed (approx. 60 C) 5 min, twice

3.Cleansing

Rub each slide separately with a soft cleaning sponge, wash thoroughly, one by one, under running tap water.

4.Wash and sterilization

Dip slides in distilled water, 3 times, 10 min (100 ml/12 slides), dry in oven (150 C) for 5 min and allow to cool.

5. Acid treatment

Dip slides in 10% HCl/70% ethanol (analytical grade), followed by DEPC-treated distilled water and then 95% ethanol.

6. Silane glue treatment

Dry in oven, 5 min at 150 C. Allow to cool. Prepare a fresh 2% solution of 3-amino propyl triethoxysilane in acetone (analytical grade). Dip slides in silane solution for 10 sec only, wash twice in 100% acetone and then in DEPC- treated distilled water.

7. Dry at 42 C overnight. Store at room temperature. Use within a month.

4.3 In situ hybridization protocol for paraffin-embedded sections on slides

using 50 mer biotinylated RNA probes
very important:
Remember to autoclave all your tools a day before your experiment (include magnetic stirring bars)
Prepare glassware in an oven (120 C).
Thaw formamide.
Note safety precautions!
4.3.1 Prehybridization
1. Wash twice in xylenes (10 min each, put the dishes into the hood and collect used xylene in

plastic bottles ) to deparaffinize.
2. Wash twice in 100% EtOH, 2 min each.
3. Then 75%, 50% and 25% EtOH in PBS + Tween-20(PBT),

(2 min each).
4. Wash twice in PBT (5 min each).
5. Treat the slices with proteinase K: insert the rack with the slides into a solution of PBT and

proteinase K(10 g/ml) and incubate 8 min at room temperature. The incubation time of this

step must be calibrated for each tissue sample. Our experience showed that 8 min is appropriate for brain tissue.

10. Apply 200l/slide of prehybridization mix, cover with Parafilm, and incubate 1 h

at 60 C.

11. Replace prehybridization mix with 200 l/slide of hybridization mix including 10

g/ml biotinylated RNA probe. It is recommended to heat the hybridization mix

for 10 min at 65 C prior to hybridization. Incubate at 52 C, overnight in a humidified chamber.

Notes:

  • PBT: PBS, 0.1% Tween-20.
  • Prehybridization mix: 50% formamide, 5 x saline-sodium citrate (SSC), pH 4.5 (use citric acid to adjust pH), 50 g/ml yeast tRNA (Boehringer Mannheim), 50 g/ml heparin (Sigma H-3393). For hybridization, add probe to 10 g/ml.
  • Steps 11 and 12 are carried out under Parafilm placed on a net in a humid chamber. To avoid dehydration of sections during hybridization, the slide box contains a 3 mm Whatman paper soaked in a solution of 50% formamide, 5 x SSC at pH 4.5.
  • All other steps are carried out in glass dishes. Use enough liquid to ensure that the sections are always fully covered during the rocking (for our dishes use 200 ml solution/dish).

4.3.2 Post-hybridization washes

(1)Wash twice with solution 1 at 60 C (30 min each).

(2)Wash twice with solution 2 at 60 C (30 min each).

(3)Wash three times with Tris-buffered saline + Tween-20 (TBST).

(4)Preblock sections with 1% skim milk in TBST for 60-90 min at room temp. You can stop at this stage by keeping the slides in blocking solution at 4 C overnight.

Notes:

Solution 1: 50% formamide, 5 x SSC, (pH 4.5), 0.5% SDS.

Solution 2: 50% formamide, 2 X SSC (pH 4.5)

10 x TBST: 100 ml = 8 g NaCl, 0.2 g KCl, 25 ml 1 M Tris.HCl (pH 7.5) (Sigma), 10 ml of 10% Tween-20. On the day of use, dilute to 1 X and add levamisol (Sigma L-9756; an alkaline phosphatase inhibitor used to suppress non-specific staining) to a final concentration of 2 mM.

1X TBST = 25 mM Tris-HCl (pH 7.5); 136 mM NaCl, 2.7 mM KCl,

0.1% Tween-20.

4.3.3 Detection of biotinylated probes with streptavidin-alkaline phosphatase (AP) conjugate

1.Dilute the streptavidin AP-conjugate (Streptavidin-alkaline phosphatase RPN 1234 from Amersham Life Science) 1:50 inTBST or ELF working solution. Incubate at room temp. for 30 min under Parafilm. This step should be calibrated for each tissue by adjusting the concentration of the streptavidin AP-conjugate.

  1. Wash 3 times in TBST buffer including 2 mM levamisol, 5 min rocking at room temp. These washes may be prolonged or be performed at 37 C if background labeling occurs.

4.3.4. Detection of alkaline phosphatase activity

Two alternative methods for detection of biotinylated RNA probes are presented in our manual.

A. Colorimetric and fluorescence detection

The Fast Red substrate (Boehringer cat. no. 1496549) creates both red color and fluorescent signal (excitation:488 nm; emission: 580  16 nm)

  1. The substrate should be prepared prior to use. Dissolve 1 tablet in 2 ml of 0.1 M Tris-HCl, pH 8.2. Shake for 1-3 min until dissolved.
  2. Distribute the solution to 1.5 ml tubes, spin in microcentrifuge for approx. 1 min at maximum speed.
  3. Transfer the supernatant to a new tube.
  4. Apply 100-150 l of Fast Red solution to each slide under Parafilm and incubate at room temperature until color develops (up to 35 min).
  5. Wash for few seconds in DDW.
  6. When red color develops (view under light microscopy) stop the reaction with stop solution (25 mM EDTA, 0.05% Triton X-100, 1 mM levamisol in PBS, pH 7.2).
  7. Incubate 10 min in stop solution.
  8. Wash few seconds in DDW.
  9. The tissue is now ready for counter staining. Alternatively, the tissue sections can be mounted under cover slips and sealed for analysis.

B. Fluorescence detection using the Enzyme Labeled Fluorescence (ELF) 6605 kit (Molecular Probes, Eugene OR, USA).

1.Add 200 l of working solutionto each slide for incubation time that varies between 4-30 min. To observe fluorescence, excite at a wavelength above 400 nm (yellow) and immediately transfer to a stop solution.

2.Stop the reaction in a solution containing 25 mM ethylenediaminetetraacetic acid (EDTA), 0.05% Triton X-100, 1 mM levamisol in PBS, pH 7.2. Incubate 10 min under aluminum foil to exclude light.

3.Fix the slices in a fixative containing 4% paraformaldehyde (PFA) in PBS pH 7.4 for 15-30 min.

4.Wash rapidly with double distilled water.

5.Keep the slides in a light protected box for photography. Use the 4,6-diamidino-2-phenylindole (DAPI) set of filters for photography of the ELF signal.

Notes:

  • All washes are performed with rocking at room temp. for 5 min and include 2 mM levamisol added to the wash solution on the day of the experiment.
  • 1X ELF wash solution is diluted on the day of use from 10X ELF wash solution. You can use 1X TBST instead of 1X ELF wash solution.
  • Add levamisol to final concentration of 2 mM.
  • ELF working solution is prepared by dilution of substrate reagent 1:20 in a

developing buffer which includes 2 mM levamisol, then dilution 1:1000 of substrate additives I and subsequently II in the working solution. Use within 10 min after filtration in a 0.22 m filter (cat. no. DGA 02025 SO from Micron Separations, Westborough, MA, USA).

5. Histological staining:

5.1 DAPI counterstaining of nuclei:

4,6-diamidino-2-phenylindole (DAPI): prepare a 1 g/ml solution of DAPI in water. Apply around 100 l/slide and cover it with Parafilm. Leave it for few minutes and wash with DDW afterwards. Be cautious in using DAPI since it is carcinogenic.

5.2 Counter staining with Giemsa's stain: prepare 1% Giemsa's stain in Giemsa buffer at pH 6.8; stain slides for 3-6 min until counterstaining is achieved; view under light microscopy the two colored reaction products -- blue Giemsa-stained nuclei and red cytoplasmic signals reflecting hybridized mRNA chains.

5.3 Hematoxylin-Eosin-Light green staining:

It is possible to counterstain adjacent slices with hematoxylin-eosin -light green staining to enable observation of nuclei and cell structures in preserved sections.

Dip in distilled water 4 times at room temperature

  1. Dip in hematoxylin for 3 min, wash in distilled water 3 times, and then under running tap water for 3 min.
  2. Dip again in distilled water 3 times
  3. Dip in alcoholic eosin for 1 min and then wash 3 times in distilled water
  4. Dip in 1% dodecamolybdophosphoric acid for 8 min and then dip directly in 0.1% Light green for not more than 1 min.
  5. Dip 3 times in 95% ethanol and then dehydrate twice in 100% isopropyl alcohol, 3 min each.
  6. Dip in a solution of xylene:isopropyl alcohol (1:1, v:v) for 3 min and then in 100% xylene three times, 3 min each.
  7. Air dry for at least 30 min.

5.4 Neuronal staining by cresyl violet

1. Prepare cresyl violet solution at pH 3.7:

cresyl fast violet 5 g

distilled water 500 ml

Let stand 16-20 h before use. (Do not filter.)

2. Deparaffinize by washing twice in xylene, 5 min each wash.

3. Wash twice in 100% ethanol, 2 min each.

4. Wash in 75%, 50%, 25% ethanol in water.

5. Wash twice in water.

6. Stain in cresyl violet solution 5 min.

7. Wash in running tap water 5 min, drain slides well.

8. Dehydrate rapidly in absolute alcohol, 3 changes.

9. Clear in xylene and mount with Entellan.

6. Appendices

6.1Materials and stock solutions for in situ hybridization

Note: all solutions should be RNase-free (by diethylpyrocarbonate (Sigma DEPC) treatment) and autoclaved.

DEPC treatment of solutions:

Add 500 l DEPC to 1000 ml of the solution; mix in a fume hood for 1 to 2 hours and then autoclave.

A.Preparation of PBS (X10)

1.3 M NaCl 296.0 g

0.07 M Na2HPO4.7 H2O 75.06 g

0.03 M NaH2PO4.H2O 16.56 g

double-distilled water to 4 l

Adjust pH to 7.4.

B.Preparation of paraformaldehyde in PBS: (must be fresh!)

This fixative is made without any NaOH to prevent basic hydrolysis of RNA.

Dissolve 40 g paraformaldehyde in 1000 ml PBS X1 (DEPC-treated).

Heat while stirring to 50-60 C.

C. SSC (X20) (Na chloride-citrate)

3 M NaCl175.30 g

0.3 M Na citrate dihydrate 88.20 g

double-distilled water to 1 liter

adjust pH to 4.5 with 4 M citric acid

1X SSC = 150 mM NaCl, 15 mM Na citrate

D.Mounting compound:

1. For hematoxylin-eosin or cresyl violet stained sections use Entellan (rapid embedding agent) from Merck

2. For fluorescence detection useImmunomount (Shandon Inc., Pittsburgh). Keep slides in a box to prevent deterioration of the fluorophore.

E.Yeast tRNA 20 mg/ml from baker yeast (Boerhinger Cat. No. 109495); 100 mg in 5 ml double-distilled water (DEPC-treated).

F.Double-deionized formamide:

Mix 50 g Amberlite MB-1 with 1 l formamide for 1.5 h while stirring. Filter and add a new amount of 50 g of Amberlite MB-1 for 30 min stirring. Filter again and the solution will be ready for use (store at -20 C).

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