Daniel:Protocols/TissueFISH

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FISH On Tissue Sections[edit]

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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]

Linnarsson lab protocol

  1. Prepare fresh paraformaldehyde 4% and keep at RT
  2. Fix sections with 4% paraformaldehyde for 15 minutes at RT
  3. Rinse sections twice with RT PBS
  4. Permeabilization-may use:
    1. Ethanol (70% EtOH, >90 minute incuabation at 4C)
    2. PBS-Tween 20
    3. PBS-TritonX-100
    4. PBS-Igepal
    5. Methanol (usually the worst)
  5. Rinse sections twice with RT PBS
  6. Prepare the wash buffer and let it sit at RT
  7. Incubate sections in wash buffer for 10 minutes
  8. Warm hybridization buffer and warm it up to 37C
  9. Prepare hybridization reaction:
    1. hybridization buffer-100 uL; probe 2-10 uL
  10. Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.

Day 2

  1. Warm up wash buffer and wash buffer with DAPI to 37C
  2. Rinse with wash buffer
  3. Add fresh was buffer and incubate for 30 min at 37C
  4. Replace with fresh wash buffer with DAPI and incubate for 30 min at 37C
  5. Wash 2 times with warm 2X SSC
  6. Mount the slide

Allen Brain/Chun Lab Protocol[edit]

Chun Lab ISH Protocol

Allen Brain Project Protocol

  • For this protocol, depc/H2O can be substituted with nfH2O*

Tissue Fixation[edit]

  1. Fix slides/sections in freshly prepared 4% paraformaldehyde/PBS depc, pH 7.2-7.4 at room temp. for 30 minutes
  2. Wash 2x in 2xSSPE for 5 minutes each
  3. Permeabilize in 0.6% Triton X-100/2xSSPEdepc for 30 minutes, room temp
  4. Wash 3 x 5 minutes in 2xSSPE
  5. Treat slides for 10 minutes/room temp in acetylation buffer
    1. Make the buffer just before use
  6. Wash 3x 5 mins 2xSSPE
  7. Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
    1. Make solutions from absolute ethanol and depc/H20*.
  8. Dry in oven at 50-65C.

At this point, you can either freeze the slides in the –80C or use immediately.

Hybridization[edit]

  1. Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
  2. Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
  3. Drop temp to 65C
  4. Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
  5. Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
  6. Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
  7. Hybridize overnight appropriate temperature
    1. VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
    2. important to keep slides in a humid environment

Post-Hybridization[edit]

Wash Version 1

  1. 2xSSPE/0.6% Triton X-100 2x 45mins. At room temp
  2. 1xHSB, 3x 30 mins at hybridization temperature
  3. Add 20 uL DAPI with Slow Fade Gold, cover with coverslip, seal with nail polish

Wash Version 2

  1. 2x 2xSSC 45 mins room temp
  2. 1x 0.2x SSC 45 mins hybridization temp
  3. 1x 0.02xSSC 45 mins hybridization temp
    1. This very stringent wash may not be necessary for some probes
  4. 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]

Original Protocol

Tissue Fixation[edit]

  1. Fix tissue pieces of an appropriate size in freshly prepared 4% formaldehyde/PBS for 4–24 h
  2. Wash the tissue in PBS, 3 × 30 min. Tissue can be stored in PBS for few days at +4°C
  3. Incubate the tissue in a sucrose series with increasing concentrations: 10% for 1 h, 20% for 1 h, and 30% for 1–24 h.
    1. The incubation time also depends on the size of the tissue piece.
    2. 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]

  1. Remove slides from freezer, let them thaw and air-dry at RT for 30–60 min
    1. 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
  2. Re-hydrate sections in 10 mM sodium citrate buffer for 5 min
  3. 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
  4. Remove the Coplin jar from the water bath and let it cool down at RT
  5. Incubate the slides in 2× SSC for 5 min
  6. Transfer slides into 50% formamide in 2× SSC and incubate for at least 1 h
    1. sections can be stored in this solution at 4°C for weeks

Hybridization[edit]

  1. Prepare glass hybridization chambers as follows (Fig. 2a, b):
    1. Cut glass strips from a coverslip (e.g., 8 × 8 mm) using a diamond cutter and a ruler;
    2. Glue glass strips parallel on two opposite borders of an intact coverslip of the same size using nail polish
    3. 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
    4. Let the nail polish completely dry before use
  2. Remove slide from the formamide and wipe the liquid from the back of the slide and from the face side of the slide
    1. Avoid marked section area
  3. Place prepared hybridization chamber above the section and carefully press it against the microscopic slide
  4. Load the probe under chamber from its open side so that probe fills in the whole space above the section (Fig. 2c, d)
  5. Seal the chamber with rubber cement
    1. It is important that rubber cement covers the entire chamber periphery or even the whole chamber (see Fig. 2e)
  6. Let the rubber cement dry at RT
    1. From this step, protect slide from light
  7. 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
  8. Place slide on a hot-block at 80°C for 5 min for the probe and section denaturation of DNA
  9. 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]

  1. Remove rubber cement together with the chamber using a pair of fine forceps
  2. Wash slides in 2× SSC at 37°C, 3 × 15 min (preferably with shaking) to remove hybridization mixture
  3. Perform stringency washes in 0.1× SSC at 60°C, 2 × 5 min to remove nonspecific hybrids
  4. Equilibrate in 2× SSC for 2 min
  5. Add DAPI, let sit for at least 30 minutes
  6. Seal the coverslip with nail polish; let sit 30 minutes
    1. Sections become fragile after the FISH; therefore, strong pressure on the coverslip should be avoided
    2. e.g., pressing the coverslip with the microscope objective when focusing often completely destroys the sample