TITLE: Single molecule FISH
AUTHORS: Thuc Nguyen, Emma Garren

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Updated 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. </div></div>

[STEPS]
?. [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.
?. [Coverslip Preparation]
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.
?. [Coverslip Preparation]
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.
?. [Coverslip Preparation]
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]
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.
?. [Tissue Sectioning]
Place the frozen OCT block with embedded brain into a -20°C cryostat for 30 minutes to equilibrate to temperature.
?. [Tissue Sectioning]
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]
Reagent Prep: 4% PFA in PBS (make fresh) and pre-chill at 4°C100% methanol pre-chill at -20°C1X PBS made with nuclease-free water
?. [Tissue Fixation and Permeabilization]
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.
?. [Tissue Fixation and Permeabilization]
Wash 3x 5 mins with PBS.
?. [Tissue Fixation and Permeabilization]
Permeabilize with 100% methanol for 10 mins at -20°C.
?. [Tissue Fixation and Permeabilization]
Remove coverslips from the methanol and let dry completely at room temperature.
?. [Tissue Fixation and Permeabilization]
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]
Reagent Prep:8% SDS in PBS (vacuum filter, store at room temperature)2X SSC (vacuum filter)SecureSeal Chambers (Grace Bio-Labs #621502)
?. [Tissue Preparation]
Remove coverslips from -80°C and place on a glass slide for support.
?. [Tissue Preparation]
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.
?. [Tissue Preparation]
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.
?. [Tissue Preparation]
Pipette 200 μl of 8% SDS into the chamber. Let incubate for 10 mins at room temperature.
?. [Tissue Preparation]
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]
Reagent Prep:Probes (take out to thaw)2X SSCDAPI (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 Imaging Buffer (store at 4°C for one month)  ABCD1ReagentAmountFinal ConcentrationNotes2Dextran sulfate1 g100 mg/ml3Nuclease-free water7.3 mlDissolve dextran sulfate in water, then add other reagents.420X SSC1 ml2X5De-ionized formamide1 ml10%6tRNA500 μl1 mg/mlSigma #1019541001. Make 20 mg/ml stock.7Ribonucleoside Vanadyl Complex100 μl2 mMNEB #S140258BSA40 μl200 μg/mlAmbion #AM2616. Stock is 50 mg/ml.9Final Volume10 ml  ABCD1ReagentAmountFinal ConcentrationNotes2Glucose0.4 g8 mg/ml3Nuclease-free water48.5 mlDissolve glucose in water, then add other reagents.41 M Tris-HCl, pH 8.01 ml20 mM55 M NaCl500 ul50 mM6Final Volume50 ml
ABCD1ReagentAmountFinal ConcentrationNotes2Dextran sulfate1 g100 mg/ml3Nuclease-free water7.3 mlDissolve dextran sulfate in water, then add other reagents.420X SSC1 ml2X5De-ionized formamide1 ml10%6tRNA500 μl1 mg/mlSigma #1019541001. Make 20 mg/ml stock.7Ribonucleoside Vanadyl Complex100 μl2 mMNEB #S140258BSA40 μl200 μg/mlAmbion #AM2616. Stock is 50 mg/ml.9Final Volume10 ml
ABCD1ReagentAmountFinal ConcentrationNotes2Glucose0.4 g8 mg/ml3Nuclease-free water48.5 mlDissolve glucose in water, then add other reagents.41 M Tris-HCl, pH 8.01 ml20 mM55 M NaCl500 ul50 mM6Final Volume50 ml
?. [Hybridization]
Equilbrate tissue section in Hybridization Buffer (without probes) by pipetting 100 μl of the buffer into the SecureSeal chamber. Incubate for 5 mins.
?. [Hybridization]
Make probe mix while tissue sections are equilibrating in HB. Dilute probes in HB to final concentration of 2 ng/μl.
?. [Hybridization]
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.
?. [Hybridization]
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.
?. [Hybridization]
Wash with Probe Wash Buffer 2 x 10 min at 37°C. Push 500 μl through  while holding the coverslip over a beaker.
?. [Hybridization]
Hold the coverslip over a beaker and pipette 500 μl of 2X SSC through the chamber. Repeat once.
?. [Hybridization]
Add enzymes to the Imaging Buffer (see table below). Pipette Imaging Buffer (with enzymes) to the SecureSeal chamber. Proceed to imaging.  ABCD1ReagentAmountFinal ConcentrationNotes2Imaging Buffer without Enzymes1 ml3250 U/ml pyranose oxidase12 μl2.4 U/mlSigma #P4234-250U48 mg/ml catalase6.25 μl50 μg/mlAbcam #ab2190925200 mM trolox in ethanol2.5 μl500 μMAdd enzymes to Imaging Buffer and use immediately. Do not store.
ABCD1ReagentAmountFinal ConcentrationNotes2Imaging Buffer without Enzymes1 ml3250 U/ml pyranose oxidase12 μl2.4 U/mlSigma #P4234-250U48 mg/ml catalase6.25 μl50 μg/mlAbcam #ab2190925200 mM trolox in ethanol2.5 μl500 μM
?. [Imaging]
Use appropriate Imaging Protocol.
?. [Probe Stripping]
Reagent Prep:2X SSC65% formamide in 2X SSC (vacuum or syringe filter)
?. [Probe Stripping]
Replace Imaging Buffer in the SecureSeal chamber with 2X SSC.Optional stopping point: Store at 4°C overnight.
?. [Probe Stripping]
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.
?. [Probe Stripping]
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.
?. [Probe Stripping]
Proceed to hybridization. See section above.