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