TITLE: iDISCO protocol for whole-mount immunostaining and volume imaging
AUTHORS: Clara Huesing, Heike Muenzberg, David Burk, Hayden Torres

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol documents how to dehydrate, immunolabel, and clear tissue in preparation for light sheet microscopy.</div></div>

[STEPS]
?. [Methanol Pretreatment]
a. Clean workspaceb. Dehydrate samples with a series of MeOH/H2O washes:         20% MeOH/ 80% H2O        40% MeOH/ 60% H2O        60% MeOH/ 40% H2O        80% MeOH/ 20% H2O        100% MeOH        100% MeOHeach wash. Place samples on rocker at room temperature during washes. *Before continuing to step 1c, transfer samples into glass vials (DCM safe) b. Incubate overnight with agitation (rocker) in 66% DCM/33% MeOH at room temperature
?. [Methanol Pretreatment]
a. Prepare plastic tubes and fill with 100% methanolb. Once the DCM/MeOH incubation is complete, transfer samples into plastic tubes made in 1a.c. Wash 2x in 100% methanol ( for each wash) at room temperature. Place on rocker during washesd. Transfer samples to a 1:5 concentration solution of 30% H2O2:100% MeOH and incubate in  fridge overnight
4 °C
?. [Methanol Pretreatment]
a. Pour out H2O2/MeOH mixture and rehydrate samples with a series of Methanol/H2O washes        80% MeOH/ 20% H2O        60% MeOH/ 40% H2O        40% MeOH/ 60% H2O        20% MeOH/ 80% H2O        100% PBS each wash. Place samples on rocker at room temperature during washes. b. Incubate samples in fresh PBS overnight on rocker, at room temperature
?. [Methanol Pretreatment]
a. Pour out PBS and wash samples 2x in PTx.2 buffer, 1hr each wash on rocker, at room temperatureb. Continue to immunolabeling (step 5).
?. [Immunolabeling]
a. Incubate samples at in permeabilization solution on rocker, for 2 days
37 °C
?. [Immunolabeling]
Pour out permeabilization solution and incubate samples at  in blocking solution on rocker, for 2 days
37 °C
?. [Immunolabeling]
a. Mix the primary antibody and primary incubation solution          Tip: Refrigerate the solution at   while you are doing step 7b.b. Pour out blocking solution that your samples are submerged inc. Transfer your samples to fresh tubes and submerge them in solution you created in 7a. d. Incubate for 1.5 - 2 weeks on rocker, at room temperature         Tip: turn tubes on their side to ensure that the samples are getting adequately “washed” with the solution
4 °C
?. [Immunolabeling]
a. Pour out the primary antibody + primary incubation solution mixtureb. Wash 5x in PTwH,  each wash on rocker, at room temperaturec. Incubate in fresh PTwH overnight on rocker, at room temperature
?. [Immunolabeling]
a. Mix the secondary antibody and secondary incubation solution b. Filter the solution you made in 9a with a 50mL syringe that has a 0.22um filter attached c. Pour out PTwH, transfer samples to fresh tubes, and pour your filtered solution over themd. Cover the samples in aluminum foil to protect from photobleachinge. Incubate samples in solution for 2 weeks on rocker, at room temperature         Tip: Turn tubes on their side to ensure that the samples are adequately getting “washed” with the solution
Note: Filtering the secondary antibody solution reduces aggregation of antibodies that results in autofluorescent puncta that appears within the tissue during imaging
?. [Immunolabeling]
a. Pour out secondary antibody + secondary incubation solution mixtureb. Wash samples 5x in PTwH,  each wash on rocker, at room temperaturec. Incubate samples in fresh PTwH overnight on rocker, at room temperature
?. [Clearing]
a. Dehydrate samples with a series of MeOH/H2O washes:         20% MeOH/ 80% H2O        40% MeOH/ 60% H2O        60% MeOH/ 40% H2O        80% MeOH/ 20% H2O        100% MeOH        100% MeOH each wash. Place samples on rocker at room temperature during washes. b. Incubate samples in 100% MeOH overnight on rocker, at room temperature
?. [Clearing]
a. Make your 66% DCM/33% MeOH mixture and pour into glass jars b. Press samples between glass slides and secure in place by wrapping wire around the sandwich (Figure 2). Use enough force to secure the sample, but do not flatten sample to a point where you are altering the physiological shape! Do not use this method on soft tissue.c. As you complete a sample, place it into its corresponding glass jar that has the 66% DCM/33% MeOH mixture, place on rockerd. Incubate samples in the 66% DCM/33% MeOH mixture for  on rocker, at room temperaturee. While samples are incubating, fill fresh glass jars with 100% DCM f. Once incubation is complete, unwrap the wire used to hold the sandwich together using two pairs of forceps – this minimizes contact with DCM as you do not want to touch DCM with only bare hands or nitrile gloves! g. As you free up a sample, place it into its corresponding jar that you have filled with 100% DCMh. Wash samples 2x in 100% DCM,  each wash, on a rocker at room temperaturei. While samples are washing, fill fresh glass jars to the top with dibenzyl ether (DBE) – this is what your sample will be suspended in for long-term storagej. After step 2h. is complete, transfer samples to their corresponding glass jars that you have filled with DBEk. Thoroughly clean workspace
Note:  the following steps describe how to “sandwich” your samples. We have found this makes larger specimens more manageable for light sheet microscopy. For example, to prevent excessive curling of the spine in our thoracic samples, we use this technique to straighten out the sample. This reduces the plane depth of the specimen, which helps with imaging larger specimens.
Note: Transferring samples from 100% DCM to DBE is time sensitive. The sample will begin to dry out very quickly once removed from 100% DCM.
Following dehydration, samples can be stored in 100% MeOH for a short period of time (roughly two days). When hydrated, samples can be stored in PBS w/azide. Clearing can also be reversed if needed.