TITLE: iDisco immunolabeling in brown adipose tissue (BAT)
AUTHORS: Seoeun Lee, Lori Zeltser

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to stain Brown Adipose Tissue using iDisco protocol and visualize the result via confocal microscope.</div></div>

[STEPS]
?. [Sample Collection]
Anesthetize the mouse, perfuse with 20ml Saline and 4% PFA in 0.1M PB
?. [Sample Collection]
Dissect BAT, carefully remove white adipose tissue surrounding the BAT. The two bilateral depots can be left connected or separated, as desired
?. [Sample Collection]
Fix in 4% PFA in 0.1M PB at 4°C, overnight with shaking, then RT 1h
?. [Sample Collection]
Wash in PBS with shaking: RT 30min x 3times
?. [Sample Pretreatment with Methanol]
Dehydrate with methanol/H2O series: 20%, 40%, 60%, 80%, 100%; at RT for 1h each with shaking
?. [Sample Pretreatment with Methanol]
Wash further with 100% methanol for 1h at RT and then chill the sample at 4°C with shaking
?. [Sample Pretreatment with Methanol]
Overnight incubation, with shaking, in 66% DCM / 33% methanol / 1% deionized water at RT
?. [Sample Pretreatment with Methanol]
Wash twice in 100% methanol at RT with shaking, and then chill the sample at 4°C
?. [Sample Pretreatment with Methanol]
Bleach in chilled fresh 5% H2O2 in methanol (1 volume 30% H2O2 to 5 volumesMeOH), overnight at 4°C with shaking
?. [Sample Pretreatment with Methanol]
Rehydrate with methanol/H2O series: 80%, 60%, 40%, 20%, PBS; 1h each at RT with shaking
?. [Sample Pretreatment with Methanol]
Wash in PTx.2 (0.2% TritonX-100 in PBS) for 1h x2 at RT with shaking
?. [Immunolabeling]
Incubate samples in Permeabilization Solution (40ml PTx.2 + 10ml DMSO + 1.15g glycine), at 37°C with shaking for 2 days
?. [Immunolabeling]
Block in Blocking Solution (42ml PT2.x + 3ml Donkey Serum + 5ml DMSO) at 37°C with shaking for 2 days
?. [Immunolabeling]
Incubate with primary antibody in PTwH (0.2% Tween-20 in PBS + 0.1% of 10mg/ml Heparin stock solution in PBS)/5%DMSO/3% Donkey Serum, at 37°C with shaking for 7 days *Validated primary antibodies- Beta 3 tubulin  (Abcam ab18207. 1:400 dilution)- Tyrosine Hydroxylase (Millipore AB152, 1:400 dilution)- Pgp9.5 (Abcam ab108986, 1:400 dilution)
?. [Immunolabeling]
Wash in PTwH for 4-5 times (1-2 hours each) until the next day at RT with shaking
?. [Immunolabeling]
Incubate with secondary antibody in PTwH/3% Donkey Serum, at 37°C with shaking for 7 days- Centrifue secondary antibody at 20000g for 10 minutes before use- Use Amber tube from this step forward- Wavelength longer than 555 (ex. AF555, AF647) is recommended
?. [Immunolabeling]
Wash in PTwH for 4-5 times (1-2 hours each) until the next day at RT with shaking
?. [Clearing]
Dehydrate with methanol/H2O series: 20%, 40%, 60%, 80%, 100%; at RT for 1h each with shaking. Ption to leave onernight at this point, is desired
?. [Clearing]
3h incubation with shaking, in 66% DCM / 33% methanol / 1% deionized water at RT
?. [Clearing]
Incubate in 100% DCM for 15 minutes twice at RT with shaking to wash the methanol
?. [Clearing]
Incubate in DiBenzyl Ether (no shaking). The tube should be filled almost completely with DBE to prevent the air from oxidizing the sample. Before imaging, invert the tube a couple of time to finish mixing the solution
?. [Imaging]
The cleared sample should be tranferred to a chamber of an appropriate size (we create one with a 3D printer), sealed on a coverglass with Kwik-sil epoxy (VWR). This chamber is then mounted on Attofluor cell chamber (Thermofisher A7816) and filled with DBE
?. [Imaging]
We typically use a Nikon Ti Eclipse inverted microscope for scanning confocal microscopy, but a resonance scanner is recommended for faster imaging