In situ hybridization of chick and mouse embryonic tissue

protocol.md

Authors: Shan Sockanathan

Abstract

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.

Introduction

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

Reagents

1. 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
  • 4L
  • 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
• B1
  • (0.1 M Tris pH 7.5, 0.15 M NaCl)
  • 1L
  • 100 ml 1 M Tris
  • 30 ml 5M NaCl
  • 870 ml H2O
• B2
  • (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
• B4
  • 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

Equipment

standard equipment for molecular biology

Procedure

DIG-label In Situ Hybridizations

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

I. Tissue section:

1. 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

1. 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

1. 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

1. 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, 3×20 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

1. Rinse slide with water.

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

Timing

3-4 days

References

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)

Author information

Shan Sockanathan, Sockanathan Lab

Correspondence to: Shan Sockanathan ([email protected])

Source: Protocol Exchange (2015) doi:10.1038/protex.2015.040. Originally published online 18 May 2015.