Mar 13, 2019
Version 3
Sequential smFISH V.3
- ZengU19 BICCN Grant1
- 1Allen Institute
- BICCN / BICAN
Protocol Citation: ZengU19 BICCN Grant 2019. Sequential smFISH. protocols.io https://dx.doi.org/10.17504/protocols.io.y6nfzde
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 and it's working
Created: March 13, 2019
Last Modified: March 13, 2019
Protocol Integer ID: 21422
Keywords: smFISH
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.
Attachments
smFISH.docx
16KB
Guidelines
Ensure that all reagents are in recombinant and RNAse-free format, as we have noticed RNA degradation in solutions that contain enzymes derived from whole organisms.
We filter every solution with a 0.2um syringe filter prior to use. This reduces background spots and dust that interfere with imaging of diffraction limited spots.
For the SDS treatment after fixation and permeabilization, be gentle when dropping SDS onto the section, as well as during washes. This treatment is relatively harsh and the tissue must be treated somewhat delicately.
Materials
STEP MATERIALS
4% PFA
PBS
PBS
PBS
2X SSC
2X SSC
65% formamide/2X SSC
Protocol materials
4% PFA
PBS
PBS
PBS
2X SSC
2X SSC
65% formamide/2X SSC
4% PFA
PBS
PBS
PBS
2X SSC
2X SSC
65% formamide/2X SSC
Safety warnings
Please refer to the SDS (Safety Data Sheet) for hazard information and safety warnings.
Avoid exposure to formamide, DAPI
Before start
Ensure all incubators and ovens are at the appropriate temperature prior to experiment.
Tissue and Sectioning
Tissue and Sectioning
10-14 um cryosections are taken from fresh-frozen tissue, which are collected on poly-lysinetreated #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.
00:05:00 RT
Fixation/Permeabilization
Fixation/Permeabilization
Post-fix sections for 15 min with 4% PFA @ 4 °C.
4% PFA
00:15:00 Post-fixing
4 °C Post-fixing
Wash with PBS (1/3)
PBS
Wash with PBS (2/3)
PBS
Wash with PBS (3/3)
PBS
Permeabilize with cold methanol at -20 C for 10 min.
-20 °C Permeabilizing
00:10:00 Permeabilizing
Air dry for 30 min in fume hood (Stopping point: store coverslips at -80C)
00:30:00 Air drying
Optional: Treat sections with 8% SDS/PBS for 10 minutes, followed by 3 – 5 rinses with PBSor 2XSSC
00:10:00
Add 2ml 2X SSC
2X SSC
2 mL 2X SSC
Hybridization
Hybridization
Pre-heat hyb oven to 37 °C
37 °C oven
Place sections in hyb buffer without probes.
Add 4 ul probe 400ul hyb buffer.
4 µL probe
400 µL hyb buffer
Note
Specific to 6-well plate format – if using perfusion chamber, this volume can be reduced.
Incubate at 37 C for 2H.
37 °C Incubation
02:00:00 Incubation
Wash
Wash
Add 2 ml wash buffer to each well.
2 mL wash buffer
Incubate at 37 C for 15 min.
37 °C Incubation
00:15:00 Incubation
Remove wash buffer.
Add 2 ml fresh wash buffer and incubate at 37 C for 15 min.
2 mL wash buffer
37 °C Incubation
00:15:00 Incubation
Replace wash buffer with fresh wash buffer + DAPI (final 5ug/mL) and incubate at 37 C for 15 min.
37 °C Incubation
00:15:00 Incubation
GLOX buffer step if performing antibody stain
Mount and image or store at 4 C in 2XSSC until imaging session
4 °C
2X SSC
Stripping
Stripping
65% formamide/2X SSC, 10 min X 3, 30 C
65% formamide/2X SSC
00:10:00
30 °C
Wash in 2XSSC (1/3)
Wash in 2XSSC (2/3)
Wash in 2XSSC (3/3)
Following stripping, proceed to hybridization step.
go to step #10 Hybridization step