Daniel:Protocols/TissueFISH: Difference between revisions

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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''.
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''.


[[Media:FISH_TissueProtocol.pdf|Original Protocol]]
==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


==Solovei Protocol==
==Solovei Protocol==
[[Media:FISH_TissueProtocol.pdf|Original Protocol]]


===Tissue Fixation===
===Tissue Fixation===

Revision as of 21:47, 27 September 2014

FISH On Tissue Sections

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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

  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

Solovei Protocol

Original Protocol

Tissue Fixation

  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

  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

  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

  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