par-seqFISH

Making solutions: 

Primary hybridization buffer (1.67x for a 50% final)

Primary and secondary wash buffers

10% wash buffer / 55% wash buffer 

Secondary hyb buffer: 10% EC (1.67x for a 10% final)

 50%(v/v) EC solution

10% (v/v) EC solution 

Imaging solution:

Anti-bleaching buffer base (high salt)

Assembling the solution:

Preparation of PDL + Bind silane coated slides:

It is recommended to treat the coverslips with plasma cleaner. However, its also possible to get good results without it.

• Place coverslips in slide holder and fill with 100% EtOH to cover the coverslips completely
• Remove access EtOH and place in oven to dry completely (about 5-10min)
• Place in 1% Bind silane for 1h RT
  • 180 ml EtOH (100%), 2ml 1M Acetic Acid, 16ml RNAse free water, 2 ml Bind Silane.
• Wash 3 times in EtOH
• Heat in oven for 30 mins 90C
• Place ~400uL of PDL solution (PDL 100µg/ml in 1X PBS) on each coverslip and let it sit O.N RT
• Remove solution gently. 
• Wash with RNAse free water twice
• Remove water and let coverslips dry (can also dry with compressed air)
• Attach coverslips to the glass-slides by placing a drop of ethanol on the slide and putting the coverslip on it (coated side up-this is where the tissue will be)
• Store in (-20) 

Acrylamide based gel coating

Preparing APS and TEMED solutions

• APS -> 0.2g in 800ul H20
• 4ul TEMED + 16ul H20
• Both on ice until use

Preparing Initiator solution

• Make 25% VA044 (Wako Chemicals) by mixing 0.125g + 400ul H20
• Can keep for a few days in 4c 

Prepare Acrylamide (AA) solution:  dilute 30% -> 4% 

• 133.3 ul of 30% Acrylamide/Bis Solution, 19:1
• 100ul 20x ssc 
• 766.6 H20 (complete to 1ml)

Degas 4% AA (remove O2)

• Make 2 holes in Eppendorf tubes using a need
• Insert needle with N2 and leave one hole for output 
• Needle directly into solution.
• Run N2 10min
• Close holes with sticker 
• Place on ice 

Prepare the gel mixture (Mix on ice)

• 958 ul 4% AA 
• 40 us VA 25% (initiator)
• 1ul APS
• 1ul TEMED

Casting the gel

• Place a 0.1 mm donut shaped sticker / chamber around your sample. You can stack a few if needed.
• Cover sample with gel mixture and place a slide to weight it down.
• Place in a tip box chamber and degas again for 15min (make 2 holes in top and insert N2)
• Close the holes
• Incubate at 37c for 1-3 hours.
• Gently remove the slide without ripping away the gel and your sample.

The reactions

Mounting cells on coverslips

• Place a drop of fixed and permeabilized bacteria (5-10ul) on the coverslip
• Let sit on bench for 5 min
• Centrifuge coverslip 500g-1000g for 5min 
• Place a hybridization chamber 

Primary probe hybridization

1. Assemble reaction mixture and mix well -> insert in to hyb chamber
2. Incubate at 37°C for ~16h (best done in a humidified chamber)
3. Gently remove hyb buffer by pipetting
4. Wash twice with 55% formamide wash buffer.
5. Fill chamber with 55% wash buffer and incubate for 30min at 37°c (or RT).
6. Wash 3 times with 2x SSC buffer.

Secondary probe hybridization 

1. Assemble reaction and add mixture to the sample.
2. Incubate at RT for 20 min in dark 
3. Wash twice with a 10% wash buffer. 
4. Fill chamber with 10% wash buffer and incubate for 5 min.
5. Wash twice with 2x SSC.
6. Optional: add DAPI for 1 min to stain cells.
7. Replace with an imaging buffer (anti-bleaching) and keep in the dark until imaging.
8. Go take some images!