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Contents

CLARITY Tissue Preparation

Method

For EDC-CLARITY, initial tissue preparation is the same as for CLARITY immunostaining. Follow Steps 1-3 of the initial CLARITY protocol, with a few modifications (included below).

Step 1: Hydrogel solution and sample preparation (<1 day)
Step 2: Hydrogel solution incubation (2-3 days)
  • Incubate sample at 4°C to let the hydrogel monomers diffuse inside the tissue, shaking optional
    • Note: Longer incubation times (1-2 weeks) will probably be needed for non-perfused tissue. Do not store samples indefinitely in the hydrogel solution as polymerization will slowly occur over time.
  • Prepare clearing solution
Step 3: Hydrogel tissue embedding (<1 day)

Acrylamide Monomer Solution

The following table summarizes the ingredients of the 1% acrylamide hydrogel solution. Amounts are listed for a 400 mL batch of solution.

Ingredient Amount Final Concentration Purpose
40% Acrylamide 10 mL 1% Hydrogel network monomer
2% Bis-acrylamide 2.5 mL 0.00625% Small chemical crosslinker
VA-044 Initiator 1 g 0.25% Polymerization thermal initiator
16% Paraformaldehyde 100 mL 4% Biomacromolecule crosslinker
10X PBS 40 mL 1X Salt buffer
Deionized water 247.5 mL - Aqueous solvent

EDC Fixation

Solution Preparation
Ingredient Amount Final Concentration Purpose
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide 0.19 g 0.1M Crosslinker
5-ethylthio-1H-tetrazole 0.13 g 0.1M Crosslinker
Methylimidizole Buffer 80 uL Buffer
Water to 10 mL

pH to 8.5

This compound acts as a fixative for 5’ terminal phosphates (Pall and Hamilton, Nature Protocols 2008). This fixative is particularly helpful for preserving and detecting small RNAs, but also improves mRNA detection.

  • Note: EDC fixation will increase clearing time by a few days.
Fixation
  • Wash tissue in methylimidizole buffer alone (80 uL in 10 mL water) to remove PBST. If PBS is incompletely washed out, it will reduce pH and cause EDC to precipitate.
  • Incubate tissue in EDC solution at 37°C overnight.
  • Transfer to clearing solution.

Lipid Removal

Clearing solution wash
  • Incubate sample in clearing solution overnight to wash out excess unreacted monomers and EDC from the tissue (37°C or room temperature, shaking preferable)
  • Replace 50 mL clearing solution and continue incubation at 37°C or room temperature
    • Note: Washes from the first 2 days contain toxic hydrogel monomers and must be disposed of as waste. Afterwards, clearing solution is safe for sink disposal.
  • Incubate sample at 37°C with shaking and continue to replace clearing solution every 1-2 days until sample is transparent (~2 weeks for 1mm sections, ~ 1 months for whole brain). The tissue will clear slowly from passive diffusion of SDS micelles.
    • Note: The duration of passive clearing is dependent on factors such as the size/thickness of the tissue sample and the incubation temperature. Clearing occurs faster for smaller, thinner samples and at higher incubation temperatures (37-50°C). Gentle shaking and routinely replacing the clearing solution may also help quicken the passive clearing process.


PBST buffer wash
  • Remove sample from the ETC chamber or clearing solution (passive clearing) when it appears see-through (sample will be swollen to a larger size and not entirely visually transparent)
  • Place sample in 50 mL PBST (0.1% TritonX in 1X PBS) at room temperature or 37°C on a shaker plate overnight to wash out SDS micelles
  • Replace PBST buffer and continue incubation with shaking overnight
    • Note: Samples can be stored indefinitely in PBST buffer. Store samples at room temperature or 4°C. The samples will remain swollen and possibly turn cloudier than they appeared in clearing solution.
    • Note: TritonX is important for washing out the SDS micelles and preventing precipitation later in the mounting solution. Do not substitute PBS for PBST when transferring the sample from clearing solution.

Probe Design and Sequences

List of validated probes and hybridization conditions
Click here to add your new probe sequence and experimental information to the database (with attribution).

Hybridization

We have had success with various types of probe design in EDC-CLARITY tissue, with hybridization conditions optimized for probe length and nucleic acid type.

Hybridization Solutions

DIG-Labeled LNAs
  • Quench endogenous peroxidase activity
    • Incubate tissue in 1% hydrogen peroxide in PBST at RT overnight
    • Wash 3 x in PBST (30min - 1h ea) at RT
Ingredient Amount Final Concentration
Formamide 5 mL 50%
20x SSC 2.5 mL 5x
Yeast tRNA 500 uL 0.5mg/ml
Water 2 mL
50mer DIG- or Initiator- Labeled Oligonucleotides

For DIG-labeled probe:

  • Incubate tissue in 1% hydrogen peroxide in PBST at RT overnight
  • Wash 3 x in PBST (30min - 1h ea) at RT

For Initiator Probes, proceed directly with hybridization:

Ingredient Amount Final Concentration
Formamide 4 mL 40%
20x SSC 1 mL 2x
Yeast tRNA 500 uL 0.5mg/ml
50% Dextran Sulfate 2 ml 10%
Water 2.5 mL
20mer “smFISH” Initiator Labeled Oligonucleotides
Ingredient Amount Final Concentration
Formamide 1 mL 10%
20x SSC 1 mL 2x
Yeast tRNA 500 uL 0.5mg/ml
50% Dextran Sulfate 2 ml 10%
Water 5.5 mL

Hybridization Protocol

Step 1: Equilibrate
  • Tissue equilibrated in hybridization solution (without probe) for 1 hour, according to the probe type to be used.
Step 2: Hybridize
  • Tissue is transferred to hybridization solution containing probe to target RNA and 10nM N50 probe to reduce non-specific binding.
  • Incubate at hybridization temperature overnight. For LNAs, we have used hybridization temperatures ~20 degrees below the Tm. For hybridization with 50mer or 20mer DNA oligonucleotides, we have performed hybridizations at 37°C. Formamide and salt concentrations were altered so that we could hybridize at 37°C to better preserve endogenous fluorescence in transgenic samples.
Step 3: Stringency

Washes performed at hybridization temperature.

  • LNAs
    • 2x 1 hour (5x SSC)
    • 1x 1 hour (1x SSC)
  • 50mers
    • 3x 1 hour (40% formamide, 2x SSC)
    • 2x 1 hour (5xSSCT)
  • 20mers
    • 3x 1 hour (10% formamide, 2x SSC)
    • 2x 1 hour (5xSSCT)

Amplification

TSA Amplification

  • Rinse in PBST at 37°C (~30min).
  • Transfer to anti-DIG-POD Fab fragment antibody (Roche) in PBST. Incubate at 37°C (1:500) overnight.
  • Wash with PBST 3 x (60 min ea) at room temperature, plus once overnight.
  • TSA reaction (Perkin Elmer, TSA Plus Fluorescein).
    • Dilute fluorescein 1:50 and incubate tissue section for 30 minutes.
  • Wash with PBST, 3 x 60 min ea. at RT.
  • Focus Clear (4h-o/n), ready for mounting/imaging.'

HCR Amplification

Step 1: Equilibrate
  • Pre-incubate in amplification buffer, 1 hour.
Ingredient Amount Final Concentration
20xSSC 10 mL 5x
Tween20 (10%) 400 uL 0.1%
50% Dextran Sulfate 4 ml 10%
Water 21.6 mL
Step 2: Prepare Hairpin Solution

For 300μl of amplification buffer (120 nM):

  • Snap cool hairpins
    • 12μl of 3μM Hairpin 1 + 4 μl of 20xSSC in PCR tube
    • 12μl of 3μM Hairpin 2 + 4μl of 20xSSC in PCR tube
    • Heat both tubes to 95°C for 90 seconds, cool to room temperature 30 minutes.
  • Add both hairpins to 300μl amplification buffer in Eppendorf tube.
    • For B1 hairpins with Alexa647, we use 120nM. For B2-Alexa543 and B5-Alexa514 hairpins we use 240nM.
  • Transfer CLARITY tissue to hairpin solution, incubate overnight at room temperature. For tissue >2mm thick, it may helpful to incubate for 2 days.
Step 3: Washes
  • Wash 5 x 1 hour in 5xSSCT at room temperature.


Refractive Index Matching

Transfer to refractive index matching solution; wait until transparent (1-4 hours). Signal is stable in FocusClear 1-2 days. In our experience, signal is stable for longer periods in ScaleA2, RIMS, or Glycerol, but sample transparency suffers, so may be suitable for <1mm sections, but not for larger volumes. Larger volumes can be challenging to make transparent again during refractive index matching. During hybridization and stringency, the tissue shrinks considerably. For tissue >2mm thick, we’ve found it helpful to re-expand the tissue before refractive index matching by transferring the tissue from 5xSSCT (after hairpin amplification) back to clearing solution (4% SDS in 0.2M borate buffer, 37 oC) overnight, then wash three times with 0.2M borate buffer to remove SDS. The expanded tissue equilibrates to RI matching in FocusClear more quickly and more thoroughly