In situ hybridization of chick and mouse embryonic tissue

doi:10.1038/protex.2015.040

Method Article

In situ hybridization of chick and mouse embryonic tissue

https://doi.org/10.1038/protex.2015.040

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The detection of transcripts in sectioned tissue by in situ hybridization is a useful method to localize sites of gene expression during development. This protocol is one that we have modified from Scharen-Wiemers and Gerfin-Moser Histochemistry 100:431-440.

Developmental biology

embryo

sections

mRNA

This protocol describes a step by step method to detect mRNA transcripts on tissue sections cut using a cryostat.

Dig Probe In vitro Transcription

H2O = 13 µl

10x transcription buffer = 2 µl

dig-NTP =2 µl

(1mg) DNA =1 µl

polymerase =1. 5 µl

RNasin =0.5 µl

2 hr 37 C

add 30 µl DEPC H20

check 2.5 µl on 1x TAE minigel (Ethidium RNA staining stronger than vector band)

spin column

add hyb mix to a total volume of 200 µl

typically 10-20 µl probe added to 1 ml Hyb mix

0.2M Phosphate Buffer (P.B) (4L)

165.3 gr Na2HPO4 7H2O (MW 268.07)

25.6 gr NaH2PO4 H20 (MW137.99)

water to 4000 ml

Note: Made with RNASE free reagents

4% paraformaldehyde (made fresh) (400ml)

200 ml water

16 gr paraformaldehyde

heat to 60-70 C, add 1 drop 10 N NaOH, and stir 10 minutes to dissolve

add 200 ml 0.2M P.B. (final conc. 0.1M)

add 3 gr NaCl

sterile filter and store on ice

PBS (4L)

(0.1 M PB and 0.15 M NaCl)

Made with RNASE free reagents

2L 0.2 M PB

35 gr NaCl

H2O to 4L

Proteinase K buffer (400ml)

(1mg/ml Prot K, 5 mM EDTA, 50 mM Tris)

400ml

5 ml 0.5 M EDTA pH8.0

20 ml 1M Tris pH7.5

20 ml 20mg/ml Prot K

H2O to 400 ml

Hybridization Solution 1L

50% formamide 500 ml of 100%

5xSSC 250 ml of 20x

5x Denharts 100 ml of 50x

250mg/ml bakers yeast RNA (Sigma R6750) 0.25 gr

500 mg/ml herring sperm DNA 0.5 gr

150 ml H2O

(0.1 M Tris pH 7.5, 0.15 M NaCl)

100 ml 1 M Tris

30 ml 5M NaCl

870 ml H2O

(B1 + 1% heat inactivated goat or sheep serum)

B3 (0.45mm filtered)

0.1 M Tris pH 9.5

0.1 M NaCl

50mM MgCl2

4.5 µl/ml NBT (75 mg/ml) (NBT from BMB no. 1383213, in 70% dimethylformamide).

3.5 µl/ml BCIP (50 mg/ml) (BCIP from BMB no. 1383221 in 100% dimethylformamide).

0.24 mg/ml levamisole

diluted in buffer B3

standard equipment for molecular biology

DIG-label In Situ Hybridizations

Adapted from Scharen-Wiemers and Gerfin-Moser Histochemistry 100:431-440.

I. Tissue section:

Fix embryo overnight at 4 C in 4% paraformaldehyde, 0.1 M PB.
Transfer embryo to 30% sucrose in 0.1M PB for 4 hr at 4 C, addition of some fixative probably reduces risk of RNase activity.
Mount in tissue tek (can store at -70 C several weeks if necessary).
Cut 12.5µm sections (thicker sections may increase signal).
Section with fisherbrand superfrost plus slides (No 12-550-15).
After sectioning air dry 20 minutes (maximum is 3 hr).

II. Tissue preparation

NOTE Eliminase treat all glassware before starting

Fix in freshly made 4 % paraformaldehyde/PBS for 10 minutes at RT.
Wash three times with PBS for 3 minutes each.
Digest in freshly diluted proteinase K (1mg/ml in 50 mM Tris 7.5, 5 mM EDTA) 5 minutes at RT. (Note: these conditions will have to be adjusted for various embryo stages.)
Fix in 4 % paraformaldehyde/PBS for 5 minutes at RT.
Wash three times with PBS for 3 minutes each.
Acetylation : 197 ml H2O, 2.4 ml triethanolamine (Fluka 90279); mix well. Add 0.52 ml acetic anhydride, and mix by dipping slides several times. Acetylate 10 minutes room temp. Start preparing mix when slides are in last wash.
Permeabilize with 1% TritonX100 in PBS for 15 minutes at room temp.
Wash 3 times with PBS for 5 minutes each

III. Hybridization

Place 500 µl hybridization buffer on slide.
Incubate at room temperature for 2 hr (overnight also works well) at room temp in a 50% formamide/5xSSC humidified chamber horizontal without coverslips.
Replace pre-hyb (pour off, dab off edge with paper towel to remove excess) with 75 µl hybridization solution containing probe at 200-400 ng/ml DIG RNA which was heated to 80 C for 5 min. and iced.
Coverslip slides, place in humidified (5xSSC, 50% Formamide) chamber overnight at 70˚C. Note: It is best to separate slides with different probes since some contamination is possible from neighboring slides in the chamber.

IV Washes/Immunological staining

Place slides in rack, submerge in 70˚C 5xSSC to remove coverslips. Carefully remove slides with forceps.
With forceps, transfer slides into 0.2XSSC at 70˚C for 1-3 hr.
Transfer to 0.2xSSC at RT for 5 min.
Transfer to buffer B1 5 minutes at RT.
Place 1-2 ml B1 with 1% heat inactivated goat (or sheep) serum (HINGS) on horizontal slides for 1 hr at RT.
Put 0.5 ml anti-DIG Ab (1:2000 dilution in B1 + 1 % HINGS) on each slide (=B2)
Place humidified chamber at 4˚C overnight (for abundant RNA 1 hr RT okay, but overnight greatly enhances signal and reduces color reaction time).
Rinse with B1 once, 3x20 minutes wash (more extensive washes may reduce background if this is a problem).
Equilibrate with B3.
Place 200 µl B4 on parafilm, and invert slide onto solution.
Incubate at RT 6 hr-3 days in humidified chamber in dark.
Stop reaction with PBS (can be kept for up to several days at 4˚C).

V. Mounting

Rinse slide with water.
Air dry completely
coverslip with Glycerol (warm to 60 ˚C, 3 drops on slide, add coverslip)

3-4 days

1.Scharen-Wiemers, N. and Gerfin-Moser, A. A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100:431-440 (1993)

The authors declare no competing financial interests

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In situ hybridization of chick and mouse embryonic tissue

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Introduction

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In situ hybridization of chick and mouse embryonic tissue

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Introduction

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Procedure

Timing

References

Additional Declarations

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Terms of Service

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