Daniel:Protocols/TissueFISH
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FISH On Tissue Sections
This page describes modified Fluorescence in Situ Hybridization (FISH) protocols for dealing with tissue samples. The protocols is taken from Fluorescence in situ Hybridization (FISH):Protocols and Applications, Methods in Molecular Biology, vol. 659, DOI 10.1007/978-1-60761-789-1_5, © Springer Science+Business Media.
Allen Brain/Chun Lab Protocol
Tissue Fixation
- Fix slides/sections in freshly prepared 4% paraformaldehyde/PBS depc, pH 7.2-7.4 at room temp. for 30 minutes
- Wash 2x in 2xSSPE for 5 minutes each
- Permeabilize in 0.6% Triton X-100/2xSSPEdepc for 30 minutes, room temp
- Wash 3 x 5 minutes in 2xSSPE
- Acetylation: Treat slides for 10 minutes/room temp. Make the buffer just before use:
- H2O/depc* 29.5ml
- Triethanolamine 0.4ml
- HCl (conc.) 52ul (microliters)
- Mix well; add 75ul acetic anhydride, stir to mix until globules are gone. Use immediately.
- Wash 3x 5 mins 2xSSPE
- Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
- Make solutions from absolute ethanol and depc/H20*.
- Dry in oven at 50-65C.
At this point, you can either freeze the slides in the –80C or use immediately.
Hybridization
- Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
- Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
- Drop temp to 65C or so Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
- Add about 25ul of probe to each slide
- if using 22x40 coverslips, 22ul for 22x30,
- Gently lower the coverslip, excluding bubbles
- Hybridize overnight in the appropriate hybridization buffer at appropriate temp
- The S1P1 and ATX5’ probes like 68-69C, most of the lpA probes 65C-
- VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
- The next day, wash the cover glasses off using a gentle spray of 2xSSC/0.1% TX100.
- Do NOT use the depc solutions-it is not necessary to be RNase free from this point on
Post-Hybridization
Wash Version 1
- 2xSSPE/0.6% Triton X-100 2x 45mins. At room temp
- 1xHSB, 3x 30 mins, 65C [TEMP VARIABLE-depends on hyb temp!)
- Wash 3 x 10 mins in 0.1M Maleic acid buffer saline MABS)
- Thoroughly removes any phosphates which might be carried over from the hybridization washes
- It is very important that no phosphates remain in the tissue or a precipitate will form in the BCIP/NBT solution!
Wash Version 2
- 2x 2xSSC 45 mins room temp
- 1x 0.2x SSC 45 mins hybridization temp
- 1x 0.02xSSC 45 mins hybridization temp
- This very stringent wash may not be necessary for some probes
- Wash 3 x 10 mins in 0.1M Maleic acid buffer saline MABS)
- Thoroughly removes any phosphates which might be carried over from the hybridization washes
- It is very important that no phosphates remain in the tissue or a precipitate will form in the BCIP/NBT solution!
Buffers
- Prehyb solution:
- 50% deionized formamide
- 50% 2xSSC pH 7.0
- Add dextran sulfate to 1%
- 20X SSPE
- NaCl 175.3g
- NaH2PO4 27.6g (anhydrous)
- EDTA/Na 7.4g
- H2O 800ml
- Maleic acid buffer/saline (MABS) for xDIG block and antibody:
- Maleic acid (mw 116.1) 11.61g
- NaCl 8.76g
- H2O 980 ml
- For this protocol, depc/H2O can be substituted with nfH2O*
Solovei Protocol
Tissue Fixation
- Fix tissue pieces of an appropriate size in freshly prepared 4% formaldehyde/PBS for 4–24 h
- Wash the tissue in PBS, 3 × 30 min. Tissue can be stored in PBS for few days at +4°C
- Incubate the tissue in a sucrose series with increasing concentrations: 10% for 1 h, 20% for 1 h, and 30% for 1–24 h.
- The incubation time also depends on the size of the tissue piece.
- Tissue can be stored in 30% sucrose at +4°C up to a few days or can be frozen in this solution for longer storage at −20°C
Pretreatment
- Remove slides from freezer, let them thaw and air-dry at RT for 30–60 min
- While the slides are drying, mark the future hybridization area as a rectangular on the back side of the slide using a diamond cutter. These marks will help to localize the hybridized area during the following procedures
- Re-hydrate sections in 10 mM sodium citrate buffer for 5 min
- Transfer slides into a Coplin jar with prewarmed (80°C) 10 mM sodium citrate buffer in a water bath and incubate there for 5–25 min
- Remove the Coplin jar from the water bath and let it cool down at RT
- Incubate the slides in 2× SSC for 5 min
- Transfer slides into 50% formamide in 2× SSC and incubate for at least 1 h
- sections can be stored in this solution at 4°C for weeks
Hybridization
- Prepare glass hybridization chambers as follows (Fig. 2a, b):
- Cut glass strips from a coverslip (e.g., 8 × 8 mm) using a diamond cutter and a ruler;
- Glue glass strips parallel on two opposite borders of an intact coverslip of the same size using nail polish
- Size of the chamber depends on a section size: chamber should fit to the section size as closely as possible in order to save DNA probe
- Let the nail polish completely dry before use
- Remove slide from the formamide and wipe the liquid from the back of the slide and from the face side of the slide
- Avoid marked section area
- Place prepared hybridization chamber above the section and carefully press it against the microscopic slide
- Load the probe under chamber from its open side so that probe fills in the whole space above the section (Fig. 2c, d)
- Seal the chamber with rubber cement
- It is important that rubber cement covers the entire chamber periphery or even the whole chamber (see Fig. 2e)
- Let the rubber cement dry at RT
- From this step, protect slide from light
- Place slide on a hot-block (or water bath) with temperature 45°C for 1–3 h in order to allow infiltration of the section with the probe
- Place slide on a hot-block at 80°C for 5 min for the probe and section denaturation of DNA
- Hybridize slide for 2 or 3 days at 37°C in a waterbath or temperature controlled oven
File:Figure2 Solovei TissueFISH.png
Post-Hybidization
- Remove rubber cement together with the chamber using a pair of fine forceps
- Wash slides in 2× SSC at 37°C, 3 × 15 min (preferably with shaking) to remove hybridization mixture
- Perform stringency washes in 0.1× SSC at 60°C, 2 × 5 min to remove nonspecific hybrids
- Equilibrate in 2× SSC for 2 min
- Add DAPI, let sit for at least 30 minutes
- Seal the coverslip with nail polish; let sit 30 minutes
- Sections become fragile after the FISH; therefore, strong pressure on the coverslip should be avoided
- e.g., pressing the coverslip with the microscope objective when focusing often completely destroys the sample