TITLE: iDISCO Clearing and Staining of Pancreas
AUTHORS: Maria Jimenez Gonzalez, Sarah  Stanley, Rosemary Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for iDISCO Clearing and immunostaining of pancreata/intrapancreatic ganglia in mice.</div></div>

[STEPS]
?. [Perfusion]
Preparation of Buffers- Heparinized PBS: 10 mg/100 ml PBS- Prepare a 4% PFA solution in PBS (Dilution of 1:4 of 16%PFA)
?. [Perfusion]
Place the solutions (Heparanized PBS and 4% PFA) in a box with ice and prime the tubes of the perfusion pump with HeparInized PBS. Anesthetize the mouse by inducing anesthesia with Isoflurane at 3%. One deeply anesthetized, place the mouse in supine position and keep anesthesia at 1-1.5% using an orofacial mask. Open the abdominal cavity, open the diaphragm and place the needle (22-25G) attached to the perfusion pump tube into the left ventricle of the heart. Subsequently, pierce the right atrium with a needle. Start the perfusion at a rate of 5 ml/min. Check that the blood is coming out and that the liver is getting pale. After infusing  aproximately, change to the infusion of 4% PFA. After 1-2 min, there must be a ‘shrinking’ of the body.After infusing of PFA, the perfusion is finished, proceed to harvest the tissues of interests in the microtubes.
20 ml
20 ml
?. [Tissue harvest]
With the use of a dissection microscope, remove the pancreas from the abdomen. Remove fat, lymph nodes, and hair as needed (easily done immersed in PBS, using dissection microscope). Keep in 4% PFA Solution at  overnight. Next day, wash 3 times with PBS before starting tissue clearing, sectioning or any other protocol. If tissue clearing, cut pancreas into approximately 8 equal pieces (to facilitate antibody permeabilization).
4 °C
?. [iDISCO - Tissue clearing]
Preparation of buffers:Ptx2 1L:- Triton X-100 2 ml- Sodium Azide 0.01%- PBS 100 mM, pH 7.4PtwH 1L:- Tween-20 2 ml- Heparin 10 mg- Sodium Azide 0.01%- PBS 100 mM, pH 7.4Permeabilization Buffer 0.5L:- Triton X-100 0.8 ml- Glycine 11.5 g- DMSO 100 ml
?. [iDISCO - Tissue clearing]
Dehydrate in an increasing gradient of Methanol/H2O, samples must be at room temperature and shaking at 20-30rpm in a wave-motion shaker:
20% MetOH for 1h
40%MetOH for 1h
60%MetOH for 1h
80%MetOH for 1h
100% MetOH for 1h
100% MetOH for 1h or overnight ()66/33% Dichloromethane (DCM)/Methanol for 3h, at room temperature, protecting samples from light100% MetOH for 1h or overnight ()
4 °C
4 °C
?. [iDISCO - Tissue clearing]
Bleaching protocol Wash twice in 100% Methanol at room temperature Chill samples at during Bleach samples in freshly prepared 5/95% H2O2/MetOH, overnight at
4 °C
4 °C
?. [iDISCO - Tissue clearing]
Rehydrate/Permeabilization in an increasing gradient of Methanol/H2O, samples must be at room temperature and shaking at 20-30rpm in a wave-motion shaker:80% MetOH for 1h
60%MetOH for 1h
40%MetOH for 1h
20%MetOH for 1hThe following are permeabilization steps:PBS for 30 minPtx2 for 1hChange the Ptx2 for 1hFinally place samples in Permeabilization Buffer for 1 day at and shaking
37 °C
?. [iDISCO - Tissue clearing]
Blocking Step:Place samples in a blocking buffer consisting of: 6%Donkey Serum , 10%Dexamethanose (DMSO) in Ptx2 for 48h at and shaking
37 °C
?. [iDISCO - Tissue clearing]
Primary Antibody Incubation:Primary antibody is diluted in 3%Donkey Serum, 10%DMSO prepared in PtwH. Samples are incubated for 96h at and shaking.  Dilution of Rabbit anti-HA is 1:500. Dilution of GP and rat anti-insulins are 1:1000.
37 °C
?. [iDISCO - Tissue clearing]
Wash in PtwH 4-5 times for 1 hour minimun per wash, shaking the samples at room temperature. Leave them in the last wash overnight.
?. [iDISCO - Tissue clearing]
Secondary Antibody Incubation:Secondary antibody is diluted in  3% Donkey Serum in PtwH. Samples are incubated for 96h at  and shaking. Dilution for all secondaries is 1:500.
37 °C
?. [iDISCO - Tissue clearing]
Wash in PtwH 4-5 times for 1 hour minimun per wash, shaking the samples at room temperature. Leave them in the last wash overnight.
?. [iDISCO - Tissue clearing]
Dehydrate in an increasing gradient of Methanol/H2O, samples must be at room temperature and shaking at 20-30rpm in a wave-motion shaker:
20% MetOH for 1h
40%MetOH for 1h
60%MetOH for 1h
80%MetOH for 1h
100% MetOH for 1h
100% MetOH for 1h or overnight
?. [iDISCO - Tissue clearing]
Clearing step:Place samples in  66/33% DCM/Methanol, 3h incubation at room temparature with shaking. Protect samples from light. Change to 100% DCM for 15 min at room temperature with shaking. Replace the 100% DCM for another 15 min incubation.Place in DBE,and shake samples at room temperature, protecting from light.  Samples should look 'transparent' after 30 min - 1 hour.  Note: clean DBE spills with MetOH. Samples can be kept in DBE for months. Better store them at
4 °C
?. [Visualization-Microscopy]
For visualization of these samples, confocal microscopy is sufficient, we use Zeiss LSM780 microscopes:- Upright microscope: Place the samples in 3D printed VisiJet samples (https://idiscodotinfo.files.wordpress.com/2015/04/whole-mount-staining-bench-protocol-methanol-dec-2016.pdf)- Inverted microscope: Place the samples in glass bottom Slides (e.g. ibidi, cat#80827)
?. [Analysis and Quantification]
Acquired images (e.g. in czi format) are converted and visualized in IMARIS software. Using the 3D Spot object quantification, we quantified the positive stained neurons and total neurons.