Daniel:Protocols/TissueFISH: Difference between revisions
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#Wash 3 x 5 minutes in 2xSSPE | #Wash 3 x 5 minutes in 2xSSPE | ||
#Acetylation: Treat slides for 10 minutes/room temp. Make the buffer just before use: | #Acetylation: Treat slides for 10 minutes/room temp. Make the buffer just before use: | ||
##H2O/depc 29.5ml | ##H2O/depc* 29.5ml | ||
##Triethanolamine 0.4ml | ##Triethanolamine 0.4ml | ||
##HCl (conc.) 52ul (microliters) | ##HCl (conc.) 52ul (microliters) | ||
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#Wash 3x 5 mins 2xSSPE | #Wash 3x 5 mins 2xSSPE | ||
#Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea. | #Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea. | ||
##Make solutions from absolute ethanol and depc/H20. | ##Make solutions from absolute ethanol and depc/H20*. | ||
#Dry in oven at 50-65C. | #Dry in oven at 50-65C. | ||
At this point, you can either freeze the slides in the –80C or use immediately. | At this point, you can either freeze the slides in the –80C or use immediately. | ||
===Hybridization=== | |||
#Prehyb slides ~ 1 hour+ at 70C with prehybridization solution | |||
Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes. I do this in a 0.5ml PCR tube in the PCR machine-after the 90C step, 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. Put into the cylindrical mailers lying flat. | |||
Hybridize overnight in the appropriate hyb at appropriate temp..-The S1P1 and ATX5’ probes like 68-69C, most of the lpA probes 65C-VARIABLE*shorter probes may want much lower. Ask Rich, Grace, or Yun-we may have already determined the right temp for the probe. If not, determine 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. | |||
Prehyb solution: | |||
50% deionized formamide | |||
50% 2xSSC pH 7.0 | |||
Add dextran sulfate to 1% | |||
Prehyb in slide mailers in the left water bath set at 75.5C (=70C) for about 2 hours. Do not dump the used prehyb in to the sink-there is a waste bottle in the hood. | |||
COOL BEFORE REMOVING SLIDES TO ADD THE PROBE!!! YOU DO NOT WANT THE SECTIONS TO DRY. | |||
*For this protocol, depc/H2O can be substituted with nfH2O | |||
==Solovei Protocol== | ==Solovei Protocol== |
Revision as of 18:32, 29 September 2014
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
- Prehyb slides ~ 1 hour+ at 70C with prehybridization solution
Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes. I do this in a 0.5ml PCR tube in the PCR machine-after the 90C step, 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. Put into the cylindrical mailers lying flat. Hybridize overnight in the appropriate hyb at appropriate temp..-The S1P1 and ATX5’ probes like 68-69C, most of the lpA probes 65C-VARIABLE*shorter probes may want much lower. Ask Rich, Grace, or Yun-we may have already determined the right temp for the probe. If not, determine 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.
Prehyb solution: 50% deionized formamide 50% 2xSSC pH 7.0 Add dextran sulfate to 1% Prehyb in slide mailers in the left water bath set at 75.5C (=70C) for about 2 hours. Do not dump the used prehyb in to the sink-there is a waste bottle in the hood. COOL BEFORE REMOVING SLIDES TO ADD THE PROBE!!! YOU DO NOT WANT THE SECTIONS TO DRY.
- 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