Dec 11, 2019

Public workspaceSingle molecule FISH

DOI
dx.doi.org/10.17504/protocols.io.xb2fiqe
  • 1Allen Institute for Brain Science
Thuc Nguyen
Open access
DOI: dx.doi.org/10.17504/protocols.io.xb2fiqe
Protocol Citation: Thuc Nguyen, Emma Garren 2019. Single molecule FISH. protocols.io https://dx.doi.org/10.17504/protocols.io.xb2fiqe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group and it is working.
Created: January 21, 2019
Last Modified: December 11, 2019
Protocol Integer ID: 19546
Keywords: single molecule FISH, fluorescent in situ hybridization
Abstract
This protocol describes multiround hybrization of directly-conjugated FISH probes for single molecule RNA detection. Thin tissue sections (10-μm) are placed onto silanized coverslips (24x50) that fit onto an ASI imaging chamber. A SecureSeal chamber is placed around the sections, which act as a reaction chamber and imaging chamber.
Materials
MATERIALS
Reagent4% PFA
ReagentCoverslip #1.5H 24x50 (Thorlabs)ThorlabsCatalog #CG15CH
Reagent3-aminopropyltriethoxysilane (APES)SigmaCatalog #A3648
ReagentASI Coverglass Imaging ChamberASI ImagingCatalog #I-3078-2450
ReagentSecureSeal Chambers 13mm x 0.8mmGrace Bio-LabsCatalog #621502

Coverslip Preparation
Coverslip Preparation
Clean coverslips (Thorlabs #CG15KH) with lens paper and 70% ethanol. With minimal handling of the coverslips (touch edges with clean gloves is ok), load them into a coverslip rack compatible with a plasma cleaning oven. We use a quartz coverslip rack. Placing coverslips in a clean glass container is ok.
Place coverslips into a plasma oven. Turn on oven. Form vacuum. Turn on plasma and let clean for 30 mins.

Pipette 100 μl of 3-aminopropyltriethoxysilane (APES, Sigma #A3648) onto a crumpled KimWipe and place next to coverslips.
Transfer coverslips to vacuum dessicator. Pipette 100 μl of 3-aminopropyltriethoxysilane (APES, Sigma #A3648) onto a crumpled KimWipe and place next to coverslips. Form vacuum and let sit for 10 min. To remove free silanes, wash coverslips by moving up and down 5 times in 100% methanol, then in fresh methanol before allowing to dry.
Store cleaned and silanized coverslips in a cool, dry, place with minimal dust.

We have used them after 2 months of storage without issues. It's possible they could last longer.
Tissue Sectioning
Tissue Sectioning
Adhere silanized coverslips to glass slides to help with picking up sections. Place 10 μl of nuclease-free water onto a clean slide and place a silanized coverslip on top. The water will hold the coverslip onto the slide during sectioning.
Place the frozen OCT block with embedded brain into a -20°C cryostat for 30 minutes to equilibrate to temperature.
Using a cryostat, make 10-μm sections and pick up on pre-chilled silanized coverslips. Let sections dry in the cryostat chamber for 30 minutes. Proceed immediately to Fixation and Permeabiliization.
Tissue Fixation and Permeabilization
Tissue Fixation and Permeabilization
Reagent Prep:
  • 4% PFA in PBS (make fresh) and pre-chill at 4°C
  • 100% methanol pre-chill at -20°C
  • 1X PBS made with nuclease-free water


Fix the tissue by placing the coverslips (attached to slides) into a slide rack and immerse in pre-chilled 4% PFA in PBS for 15 minutes at 4°C.

The coverslips will detach from the slides at this point. Let fix for 15 mins and then remove the coverslips (without slides) and into a coverslip rack for washing.
Wash 3x 5 mins with PBS.
Permeabilize with 100% methanol for 10 mins at -20°C.
Remove coverslips from the methanol and let dry completely at room temperature.
Box the coverslips into experimental sets. For example, 2-6 coverslips per box. Place each box into a zip lock bag. Seal tight. Store at -80°C.

When ready to use, remove each box and use all coverslips. Do not place back into freezer once the zip lock bag and box is opened. We have used stored tissue slices up to three weeks without noticeable issues.
Tissue Preparation
Tissue Preparation
Reagent Prep:
  • 8% SDS in PBS (vacuum filter, store at room temperature)
  • 2X SSC (vacuum filter)
  • SecureSeal Chambers (Grace Bio-Labs #621502)
Remove coverslips from -80°C and place on a glass slide for support.
Trim SecureSeal chambers so they fit within the open footprint of the ASI Imaging Chamber or equivalent, and place it around each section. It is important that all handling and room temperature steps from this point forward be performed in a clean PCR hood maintained as an RNase-free area. Samples should be covered, either in a humidified chamber or with port seal stickers, before transfer to the 37°C oven, 4°C fridge or microscope for imaging.
Pipette 2X SSC into the chamber slowly, making sure to push out the air and not introduce air bubbles. Let tissue section equilibrate to room temperature for 1 min.

Take note of how much volume is needed to fill the chamber. It is 100 μl for the chambers specified in this protocol. For reagents that need to be at a specific concentration, we typically push 200 μl through the chamber to make sure the last 100 μl is at the desired concentration.
Pipette 200 μl of 8% SDS into the chamber. Let incubate for 10 mins at room temperature.
Holding the coverslip sideways over a beaker, pipette 500 μl of 2X SSC through the chamber and let the buffer flow out the other port and fall into the beaker. Repeat 4 more times or until the output buffer is no longer soapy. All solutions should be added as slowly as possible.
Hybridization
Hybridization
Reagent Prep:
  • Probes (take out to thaw)
  • 2X SSC
  • DAPI (5 μg/ml in 2X SSC)
  • Hybridization Buffer: 10% formamide in 2X SSC with tRNA, RVC, and BSA (make aliquots and store at -20°C for up to three months)
  • Probe Wash Buffer: 20% formamide in 2X SSC

ReagentAmountFinal ConcentrationNotes
Dextran sulfate1 g100 mg/ml
Nuclease-free water7.3 mlDissolve dextran sulfate in water, then add other reagents.
20X SSC1 ml2X
De-ionized formamide1 ml10%
tRNA500 μl1 mg/mlSigma #1019541001. Make 20 mg/ml stock.
Ribonucleoside Vanadyl Complex100 μl2 mMNEB #S14025
BSA40 μl200 μg/mlAmbion #AM2616. Stock is 50 mg/ml.
Final Volume10 ml
Hybridization Buffer

  • Imaging Buffer (store at 4°C for one month)

ReagentAmountFinal ConcentrationNotes
Glucose0.4 g8 mg/ml
Nuclease-free water48.5 mlDissolve glucose in water, then add other reagents.
1 M Tris-HCl, pH 8.01 ml20 mM
5 M NaCl500 ul50 mM
Final Volume50 ml
Imaging Buffer without Enzymes

Equilbrate tissue section in Hybridization Buffer (without probes) by pipetting 100 μl of the buffer into the SecureSeal chamber. Incubate for 5 mins.
Make probe mix while tissue sections are equilibrating in HB. Dilute probes in HB to final concentration of 2 ng/μl.
Pipette the probe mix into the SecureSeal chamber. Cover ports with the port sealing stickers included with the SecureSeal pack. Place into a humified chamber and incubate for 2 hours at 37°C.

Humidified chamber can be an empty pipette tip box. Place several wet paper towels at the bottom of the box. Replace the tip rack and place the coverslips on the rack. Cover with a lid and seal with Parafilm.
Wash with Probe Wash Buffer containing 5 μg/ml DAPI for 10 mins at 37°C. Push 500 μl through while holding the coverslip over a beaker.
Wash with Probe Wash Buffer 2 x 10 min at 37°C. Push 500 μl through while holding the coverslip over a beaker.
Hold the coverslip over a beaker and pipette 500 μl of 2X SSC through the chamber. Repeat once.
Add enzymes to the Imaging Buffer (see table below). Pipette Imaging Buffer (with enzymes) to the SecureSeal chamber. Proceed to imaging.

ReagentAmountFinal ConcentrationNotes
Imaging Buffer without Enzymes1 ml
250 U/ml pyranose oxidase12 μl2.4 U/mlSigma #P4234-250U
8 mg/ml catalase6.25 μl50 μg/mlAbcam #ab219092
200 mM trolox in ethanol2.5 μl500 μM
Imaging Buffer with Enzymes

Add enzymes to Imaging Buffer and use immediately. Do not store.

Imaging
Imaging
Use appropriate Imaging Protocol.
Probe Stripping
Probe Stripping
Reagent Prep:
  • 2X SSC
  • 65% formamide in 2X SSC (vacuum or syringe filter)
Replace Imaging Buffer in the SecureSeal chamber with 2X SSC.

Optional stopping point: Store at 4°C overnight.
Add 65% formamide in 2X SSC to the SecureSeal chamber for 3x 10 mins at 37°C.

We performed this step at 37°C for this protocol. However, 30°C has also been used with success.
Wash with 2X SSC 3x 5 mins at room temperature by pipetting 500 μl through the chamber each time.

Optional stopping point: Store at 4°C overnight.
Proceed to hybridization. See section above.