Daniel:Protocols/TissueFISH
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FISH On Tissue Sections[edit]
This page describes modified Fluorescence in Situ Hybridization (FISH) protocols for dealing with tissue samples. The protocol 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.
Linnarsson Lab Protocol[edit]
- Prepare fresh paraformaldehyde 4% and keep at RT
- Fix sections with 4% paraformaldehyde for 15 minutes at RT
- Rinse sections twice with RT PBS
- Permeabilization-may use:
- Ethanol (70% EtOH, >90 minute incuabation at 4C)
- PBS-Tween 20
- PBS-TritonX-100
- PBS-Igepal
- Methanol (usually the worst)
- Rinse sections twice with RT PBS
- Prepare the wash buffer and let it sit at RT
- Incubate sections in wash buffer for 10 minutes
- Warm hybridization buffer and warm it up to 37C
- Prepare hybridization reaction:
- hybridization buffer-100 uL; probe 2-10 uL
- Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.
Day 2
- Warm up wash buffer and wash buffer with DAPI to 37C
- Rinse with wash buffer
- Add fresh was buffer and incubate for 30 min at 37C
- Replace with fresh wash buffer with DAPI and incubate for 30 min at 37C
- Wash 2 times with warm 2X SSC
- Mount the slide
Allen Brain/Chun Lab Protocol[edit]
- For this protocol, depc/H2O can be substituted with nfH2O*
Tissue Fixation[edit]
- 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
- Treat slides for 10 minutes/room temp in acetylation buffer
- Make the buffer just before use
- 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[edit]
- 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
- Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
- Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
- Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
- Hybridize overnight appropriate temperature
- VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
- important to keep slides in a humid environment
Post-Hybridization[edit]
Wash Version 1
- 2xSSPE/0.6% Triton X-100 2x 45mins. At room temp
- 1xHSB, 3x 30 mins at hybridization temperature
- Add 20 uL DAPI with Slow Fade Gold, cover with coverslip, seal with nail polish
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
- Add 20 uL DAPI with Slow Fade Gold, cover with coverslip, seal with nail polish
Buffers[edit]
- 20X SSPE
- NaCl 175.3g
- NaH2PO4 27.6g (anhydrous)
- EDTA/Na 7.4g
- H2O 800ml
- Prehybridization solution:
- 50% deionized formamide
- 50% 2xSSC pH 7.0
- Add dextran sulfate to 1%
- Hybridization solution
- 2X SSC
- 0.1% Tween-20
- 20% Formamide
- 10% Dextran Sulfate
- Acetylation Buffer; 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.
- High Stringency Buffer:
- H2O 90ml
- 0.02M Na4P2O7 10ml
- 0.2M NaH2PO4 1.5ml
- Depc/H2O:
- Add 0.05% depc to MQ H2O (500ul/liter).
- Cap tightly, mix well by shaking, then loosen cap and allow to sit overnight. Solution will outgas.
- Autoclave on program #3/liquid in a surrounding water bath (~2”depth).
Solovei Protocol[edit]
Tissue Fixation[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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