Aug 21, 2020
7: User-friendly protocol: Retina Tissue Sections RNA FISH
This protocol is a draft, published without a DOI.
- Jocelyn Y. Kishi1,2,3,
- Sylvain W. Lapan4,3,
- Brian J Beliveau1,2,5,3,6,
- Emma R. West4,3,
- Allen Zhu1,2,
- Hiroshi M. Sasaki1,2,
- Sinem K Saka1,2,
- Yu Wang1,2,
- Constance L Cepko4,7,6,
- Peng Yin1,2,6
- 1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA;
- 2Department of Systems Biology, Harvard Medical School, Boston, MA, USA;
- 3These authors contributed equally;
- 4Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA;
- 5Present address: Department of Genome Sciences, University of Washington, Seattle, WA, USA;
- 6Correspondence: py@hms.harvard.edu (P.Y.), cepko@genetics.med.harvard.edu (C.L.C.), beliveau@uw.edu (B. J. B.);
- 7Howard Hughes Medical Institute, Chevy Chase, MD, USA
- Human Cell Atlas Method Development Community
External link: http://saber.fish/
Protocol Citation: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem K Saka, Yu Wang, Constance L Cepko, Peng Yin 2020. 7: User-friendly protocol: Retina Tissue Sections RNA FISH. protocols.io https://protocols.io/view/7-user-friendly-protocol-retina-tissue-sections-rn-bh9wj97e
Manuscript citation:
Kishi, J.Y., Lapan, S.W., Beliveau, B.J. et al. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat Methods 16, 533–544 (2019). https://doi.org/10.1038/s41592-019-0404-0
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
Created: July 06, 2020
Last Modified: August 21, 2020
Protocol Integer ID: 38934
Keywords: retina tissue, RNA , fish, tissue sections, SABER, SABER-FISH,
Abstract
This protocol is about SABER RNA FISH with retina tissue.
Note
Notes: This stepwise protocol corresponds to the most rapid version we have tested for 40µm sections. Experiments in the manuscript employ pretreatment steps and longer wash and development steps that we determined to be inessential in a test involving two 500 nt probes (see Fig. S3, protocol 6).
Note
Attachments
Guidelines
Fig. S3 demonstrates that the protocol is robust to certain variations. Tissues can be treated with methanol. Hyb1 and Hyb2 incubation time can be increased, and fluorescent oligo can be applied at a range of concentrations. To reduce viscosity for ease of solution exchange, dextran sulfate can be eliminated from Hyb2. Probes can be purified using ethanol precipitation; however, this resulted in a mild decrease in signal for unknown reasons.
Branching is performed similarly to primary probe incubation, in Hyb1 solution. Branches are applied after primary probe washes are complete, and before fluorescent detection. Branches are extended to length of ~350-500 nt and incubated for at least 5 hours (longer may be required, depending on tissue penetration) in 40% formamide Hyb solution. Hyb temperature must be adjusted depending on the branches being used. We recommend using a temperature at least 1 degree lower than the Tm of the branch with the lowest melting temperature (see Fig. S2). Note that formamide concentration can be adjusted instead of oven temperature. Another option is to extend the branch annealing portion (the 30mer) by adding an additional full or partial repeat (primer sequence + T) to match the Tm of the branch with highest Tm. If doing serial multiplexing with branching, 30mers with lower melting temperatures may be partially displaced by displacement of the fluorescent oligos. For this reason we recommend using a branch with high Tm (e.g. 27*27*27*) and extending other branches as needed to match this melting temperature.
Materials
MATERIALS
Tween-20Sigma AldrichCatalog #P9416
µ-Slide 8 Well Glass BottomIbidiCatalog #80827
UltraPure™ SSC, 20XThermo FisherCatalog #15557044
Pierce™ 20X Borate BufferThermo FisherCatalog #28341
Pierce™ 16% Formaldehyde (w/v), Methanol-freeThermo FisherCatalog #28908
PBSGibco - Thermo FischerCatalog # 10010023
Poly-D-lysine hydrobromide (PDL)Sigma AldrichCatalog #P7886
ddH2O
Formamide DeionizedMerck MilliporeCatalog #S4117
Glycerol
Propyl gallate
Dextran sulfate sodium saltSigma AldrichCatalog #D8906
Solution preaparation:
Note
ddH2O is used for all solutions, not DEPC. We recommend using sterile plastic tubes and not reusable glassware for solutions. Formamide and formamide-containing solutions are aliquoted and stored at -20 °C .
Whyb (Wash hyb):
- 2 X SSC
- 1 % Tween-20
- 40 % Formamide
Example mix for 40% formamide Whyb: 1 mL 20×SSC , 1 mL 10% Tween , 4 mL Formamide (for 40% mix) , 4 mL H2O .
Hyb 1 (for primary probe hyb):
- 2 X SSC
- 1 % Tween-20
- 40 % Formamide
- 10 % Dextran sulfate
Note
Probes are generally used at a concentration of 1µg /120 µL volume
Example Hyb1 master mix: 1 mL 20xSSC , 1 mL 10% tween , 4 mL Formamide , 2 mL Dextran sulfate (50% solution) .
Example Hyb1/Probe mix: 96 µL Hyb1 master mix , 5 µL 200ng/ µL probe 1 , 5 µL 200ng/ µL probe 2 , 14 µL ddH2O .
Note
Notes: Stock of Hyb1 should be made accounting for the fact that probes, which are eluted in ddH2O, have a water volume. The example Hyb1 mix above allows up to 24 µL probe per 96 µL Hyb mix for a total of 120 µL volume to add to the well. For higher multiplexing probes can also be concentrated to reduce water volume by using a SpeedVac or heat block. After addition of probe to Hyb1 mix, the solution should be mixed well by pipetting until uniform consistency is observed. Likewise, mix well by rocking after adding dextran sulfate to the Hyb1 mix. Dextran sulfate is usually added from 50 % (w/v) stock made by dissolving powder in ddH2O. Dextran sulfate takes time to dissolve, and volume will shrink as powder dissolves. Use tick marks on the side of the tube to add water to final volume after most of the powder has dissolved.
Hyb 2 (for fluorescent detection):
- 1 X PBS
- 0.2 % Tween-20
- 10 % Dextran sulfate
Fluorescent oligos added to concentration of 0.2 micromolar (µM) -1 micromolar (µM) .
Example Hyb2 master mix: 1 mL 10×PBS , 200 µL 10% Tween-20 , 2 mL dextran sulfate , 4.8 mL H2O..
Example Fluor/Hyb2 mix: 96 µL Hyb2 master mix , 2.4 µL Fluor Oligo 1 (10µM) , 2.4 µL Fluor Oligo 2 (10µM) , 19.2 µL ddH2O .
Note
Notes: Master mix aliquots are stored at 4 °C . Side-by-side testing with two probes indicates 0.2 micromolar (µM) fluorescent oligo is sufficient. Dextran sulfate can be excluded from the Hyb2 mixture (tested for fluorescent oligo at 1 micromolar (µM) ).
Displacement buffer:
- 1 X PBS
- 50 % formamide
Stored at -20 °C .
Example master mix: 1 mL 10×PBS , 5 mL Formamide , 4 mL H2O .
Glycerol mounting media:
- 50 % glycerol
- 1 X PBS
- 20 millimolar (mM) Tris, pH 8
- 2.5 mg/ml propyl gallate
Example mix: 8 mL 100% glycerol , 1 mL 10×PBS , 200 µL 1M Tris , 25 mg propyl gallate
Stored at 4 °C .
Note
Note: Resuspend fully and spin in centrifuge before use (3 minutes on max in table top centrifuge) to remove undissolved propyl gallate specks that fluoresce. This mounting media appears to have a shelf life of 6 months when stored properly and appears to degrade signal beyond this point.
PBSTw:
Mg/Ca-free, RNAse/DNAse free PBS (Gibco #10010-023) with 0.1 % Tween-20 .
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Note
Note: After rehydrating sections, do not allow them to dry completely at any point in the protocol. Washes should be performed such that sections do not remain devoid of solution for more than a few seconds. For washes done at elevated temperature, solutions should be prewarmed and added in or near the oven. Avoid letting samples cool during these wash steps. Volumes used are 120 µL for Hyb1 (minimal to cover tissue), and 150-200 µL for all other steps. When pipetting the hyb mixes with dextran sulfate, allow time for the viscous solution to fully accumulate in the pipette tip to avoid loss of volume.
Fixation
Fixation
Dissect neural retinas in PBS and fix in 4 % FA (diluted from 16 % FA ampule , Thermo Fisher #28908) for 00:25:00 at Room temperature .
Embed retinas in a 1:1 (v/v) mixture of 30 % sucrose (in 1×PBS) and OCT, frozen in ethanol bath (200 proof ethanol cooled in dry ice). Be careful to ensure that ethanol bath does not contact the embedded retina or the embedding solution.`
Store frozen blocks at -80 °C prior to sectioning.
Preparing the slide
Preparing the slide
Prepare PDL using 0.3 mg/ml PDL (sigma P7886) dissolved in 2 X Borate buffer , diluted from 20 X stock (Thermo Scientific, #28341) with ddH2O.
Store PDL in aliquots at -20 °C .
Apply PDL 8-well Ibidi chamber slide (Cat #80827) for at least 00:30:00 .
Then remove PDL.
Dry the slide.
Wash once in ddH2O.
Dry again.
Sectioning
Sectioning
Place the PDL coated and fully dried slide inside the cryostat ahead of sectioning. Do not remove slide until all sections are in place or condensation will form that can impede adhesion.
Place sections inside wells using brushes, and flatten sections as much as possible to prevent folding when the slide is transferred to Room temperature .
After removing the slide from the chamber, spin at 600 x g for 00:03:00 (plate spinner centrifuge) 1-2 times to promote adhesion.
Hybridization
Hybridization
Wash sections 3×00:05:00 in PBSTw to remove OCT.
Wash sections 00:05:00 in PBSTw to remove OCT. (1/3)
Wash sections 00:05:00 in PBSTw to remove OCT. (2/3)
Wash sections 00:05:00 in PBSTw to remove OCT. (3/3)
Replace PBSTw with Whyb.
Place in hybridization oven set to 43 °C , minimum 00:10:00 .
In oven, set to 43°C
In oven, set to 43°C
Replace Whyb with Hyb1/Probe mixture pre-warmed to 43 °C . Seal chamber with parafilm. Fill unused wells with Whyb to maintain humidity in the chamber sli.
Incubate 16:00:00 minimum.
Replace Hyb1 with Whyb (quick wash to remove residual Hyb1).
Wash 2×00:30:00 in Whyb.
Wash 00:30:00 in Whyb. (1/2)
Wash 00:30:00 in Whyb. (2/2)
Wash 2×00:05:00 in 2 X SSCT .
Wash 00:05:00 in 2 X SSCT . (1/2)
Wash 00:05:00 in 2 X SSCT . (2/2)
Return to Room temperature (Pause point: sample can be stored in 2 X SSCT or PBSTw at 4 °C for several weeks).
Fluorescent detection
Fluorescent detection
Replace 2 X SSCTw with PBSTw (2 washes at Room temperature ).
Set oven to 37 °C .
Transfer slide to 37 °C for hybridization and subsequent wash steps.
Once slide is warm, remove PBSTw and add Hyb2/fluor solution (prewarmed).
Incubate at least 00:10:00 at 37 °C .
Replace Hyb2 with PBSTw (prewarmed, quick wash).
Wash 2×00:05:00 in PBSTw.
Wash 00:05:00 in PBSTw. (1/2)
Return to Room temperature (Pause point: Samples can be stored at 4 °C without obvious signal loss for at least 1 week).
Imaging
Imaging
Wash once in PBS and add mounting media until sections are covered (See below for notes on imaging).
Serial detection (complete before repeating fluorescent detection)
Serial detection (complete before repeating fluorescent detection)
Remove glycerol mounting media with 3 or 4 PBSTw washes (~00:05:00 ).
Wash PBSTw. (1/4)
Wash PBSTw. (2/4)
Wash PBSTw. (3/4)
Wash PBSTw. (4/4)
Wash 3×00:05:00 in Displacement buffer at Room temperature .
Wash 00:05:00 in Displacement buffer at Room temperature . (1/3)
Wash 00:05:00 in Displacement buffer at Room temperature . (2/3)
Wash 00:05:00 in Displacement buffer at Room temperature . (3/3)
Wash 3×00:02:00 in PBSTw.
Wash 00:02:00 in PBSTw. (1/3)
Wash 00:02:00 in PBSTw. (2/3)
Wash 00:02:00 in PBSTw. (3/3)