Table 1

Table of step-by-step instructions for our modified version of the iDISCO+ immunolabelling and tissue clearing protocol for staining Fos in intact mouse brains

Step
#
ChemicalTimeTemperatureContainerNotes
1aFirst PBS, then 4% PFA---Transcardial perfusion
1b4% PFAOvernight4°C15-ml Falcon tube
1cPBS30–60 min × 3 timesRT15-ml Falcon tubeMix using a rotary shaker
Note: Prior to the next step, samples can be transferred to PBS with 0.1% sodium azide and stored long
term at 4°C if necessary
2a100% dH2O1 h × 2RT15-ml Falcon tubeMix using a rotary shaker
2b20% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
2c40% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
2d60% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
2e80% MeOH in dH2O1.5 hRT15 ml Falcon tubeMix using a rotary shaker
2f100% MeOH1.5 hRT15-ml Falcon tubeMix using a rotary shaker
2g100% MeOHOvernightRT15-ml Falcon tubeMix using a rotary shaker
3a66% DCM + 33% MeOH2 hRT15-ml Falcon tubeMix using a rotary shaker and wait till the brain sinks to the bottom
3b66% DCM + 33% MeOHOvernightRT15-ml Falcon tubeMix using a rotary shaker and wait till the brain sinks to the bottom
3c100% MeOH3 hRT15-ml Falcon tubeMix using a rotary shaker
4a100% MeOH3 hRT15-ml Falcon tubeMix using a rotary shaker
4b5% H2O2 in 100% MeOHOvernight4°C15-ml Falcon tubeMix using a rotary shaker
4c80% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
4d60% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
4e40% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
4f20% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
4g100% dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
5aPBS1 hRT15-ml Falcon tubeMix using a rotary shaker
5bPTx0.5 (PBS with 0.5% Triton X-100)1 h37°C15-ml Falcon tubeMix using a rotary shaker
5cPTx0.51 h37°C15-ml Falcon tubeMix using a rotary shaker
5dPTx0.5Overnight37°C15-ml Falcon tubeMix using a rotary shaker
Note: Prior to the next step, samples can be transferred to PTx0.5 with 0.1% sodium azide and stored for a few
days at 37°C if necessary
6aPermeabilization buffer (78.6% PTx0.5 + 1.4% glycine + 20% DMSO)48 h37°C15-ml Falcon tubeMix using a rotary shaker; incubation time can be extended by 24–48 h if needed
6bBlocking buffer (84% PTx0.5 + 6% NDS + 10% DMSO)48 h37°C15-ml Falcon tubeMix using a rotary shaker; incubation time can be extended by 24–48 h if needed
Note: For the next step, we observed that five sequential additions of 1° antibody at 1:1000 dilution (1° Ab booster) helped
improve penetration and labeling over a single bolus addition at a 1:200 dilution
7a1° Antibody (Ab) buffer (92% PTwH0.5 + 3% NDS + 5% DMSO) with 1:1000 1° Ab24 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
7b1° Ab booster (1:1000 addition of 1° Ab)24 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
7c1° Ab booster24 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
7d1° Ab booster24 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
7e1° Ab booster72 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
8aPTwH0.5 (PBS with 0.5% Tween 20 + 10 μg/ml heparin)24 h37°C15-ml Falcon tubeMix using a rotary shaker
8bPTwH0.524 h37°C15-ml Falcon tubeMix using a rotary shaker
8cPTwH0.524 h37°C15-ml Falcon tubeMix using a rotary shaker
8dPTwH0.524 h37°C15-ml Falcon tubeMix using a rotary shaker
Note: Prior to the next step, samples can be transferred to PTwH0.5 with 0.1% sodium azide and stored for a few days
at 37°C if necessary; for the next step, we observed that five sequential additions of 2° antibody at 1:500 dilution (2° Ab
booster) helped improve penetration and labeling over a single bolus addition at a 1:100 dilution
9a2° Ab buffer (97% PTwH0.5 + 3% NDS) with 1:500 2° Ab24 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
9b2° Ab booster (1:500 addition of 2° Ab)24 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
9c2° Ab booster24 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
9d2° Ab booster24 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
9e2° Ab booster48 h37°C1.2-ml Corning cryovial with external threadsMix using a rotary shaker
10aPTwH0.524 h37°C15-ml Falcon tubeMix using a rotary shaker
10bPTwH0.524 h37°C15-ml Falcon tubeMix using a rotary shaker
10cPTwH0.524 h37°C15-ml Falcon tubeMix using a rotary shaker
10dPTwH0.524 h37°C15-ml Falcon tubeMix using a rotary shaker
Note: Prior to the next step, samples can be transferred to PBS with 0.1% sodium azide and stored long
term at 4°C if necessary
11a100% dH2O1 hRT15-ml Falcon tubeMix using a rotary shaker
11b20% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
11c40% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
11d60% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
11e80% MeOH in dH2O1.5 hRT15-ml Falcon tubeMix using a rotary shaker
11f100% MeOH1.5 hRT15-ml Falcon tubeMix using a rotary shaker
12a100% MeOH1.5 hRT15-ml Falcon tubeMix using a rotary shaker
12b66% DCM + 33% MeOH2 hRT15-ml Falcon tubeMix using a rotary shaker and wait till the brain sinks to the bottom
12c66% DCM + 33% MeOH2 hRT15-ml Falcon tubeMix using a rotary shaker and wait till the brain sinks to the bottom
12d66% DCM + 33% MeOHOvernightRT15-ml Falcon tubeMix using a rotary shaker and wait till the brain sinks to the bottom
12e100% DCM3 hRTWide mouth amber vial with PTFE lined capMix using a rotary shaker and wait till the brain sinks to the bottom
12f100% DCM3 hRTWide mouth amber vial with PTFE lined capMix using a rotary shaker and wait till the brain sinks to the bottom
13aDBE3 hRTWide mouth amber vial with PTFE lined capMix using a rotary shaker and wait till the brain sinks to the bottom
13bDBE3 hRTWide mouth amber vial with PTFE lined capMix using a rotary shaker and wait till the brain sinks to the bottom
13cDBE3 hRTWide mouth amber vial with PTFE lined capKeep stationary
14Intact brain imaging using LSFM
  • Times recommended for each step are listed along with sample preparation details and conditions.