Sequential smFISH Allen Institute
Jennie Close, Zoe Maltzer
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Abstract
We have developed a multiplexed single molecule FISH protocol for use at the Institute. This protocol was optimized on human tissue, but will work on mouse tissue as well. It was adapted from Lyubimova et. al., Nature Protocols, 2013.
Before start
Ensure all incubators and ovens are at the appropriate temperature prior to experiment.
Attachments
Steps
Tissue and Sectioning
10-14 um cryosections are taken from fresh-frozen tissue, which are collected on poly-lysine-treated #1 coverslips at room temperature (RT). After 5-10 min at RT, sections are placed at 4°C until sectioning is complete. At that point, proceed immediately to fixation and permeabilization.
Fixation/Permeabilization
Post-fix sections for 15 min with 4% PFA @ 4C
Wash with PBS 3X
Permeabilize with room temperature isopropanol 3 min
Air dry for 30 min in fume hood (Stopping point: store coverslips at -80C)
Optional: Treat sections with 8% SDS/PBS for 10 minutes, followed by 3 – 5 rinses with PBSor 2XSSC
Add 2ml 2X SSC
Hybridization
Place sections in hyb buffer without probes for 5 min.
Add probes to 400ul hyb buffer at a final concentration of 2ng/ul* (specific to 6-well plate format – if using perfusion chamber, this volume can be reduced)
*We store a working 200ng/ul stock of probes in the dark at 4C. These are diluted 1:100 for hybridizations, but this may need to be adjusted depending on the probe.
Incubate at 37 C for 2H
Wash
Add 2 ml wash buffer to each well, incubate at 37 C for 15 min
Remove wash buffer
Add 2 ml fresh wash buffer and incubate at 37 C for 15 min
Replace wash buffer with fresh wash buffer + DAPI (final 5ug/mL) and incubate at 37 C for 15 min
(GLOX buffer step if performing antibody stain)
Mount and image or store at 4 C in 2XSSC until imaging session
Stripping
65% formamide/2X SSC, 10 min X 3, 30 C
3 washes in 2XSSC
Imaging
Add enzymes to Imaging Buffer just prior to imaging.
