TITLE: LightsheetSampleProcessing-EmbryonicBrain
AUTHORS: Nagham Khouri-Farah

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>An imropved method for Lightsheet imaging sample preparation. We use this protocol for embryonic brain tissue (the embryonic cerebellum in particular), it could also be optimized for other types of tissues with few modifications to incubation periods. First, we fix the tissue for two hours on ice, then we pereamibilize using a mixture of Triton and Tween.</span><span style = "font-weight:bold;"> An added ingenious step </span><span>is steaming the tissue in Citric acid to retrieve epitopes after crosslinking and bleach the autofluorescence of tissue and blood vessels. That simple step saves a lot of time avoiding H</span><span style = "vertical-align:sub;">2</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">2</span><span> bleaching steps in other staining protocols and improves the quality of antibody staining. After staining, the tissue clearing is based in concept on RTF method  (Yu et al. 2018) with modifications and added incubation steps in increasing glycerol concentrations. The final imaging solution of 80% glycerol should already have a refractive index of 1.45 matching that of Zeiss lightsheet Z1 X5 lens, thus, it can be used to fill the lightsheet chamber for imaging and for sample storage.</span></div></div>

[STEPS]
?. [Tissue Fixation ]
Dissect the embryonic brain tissue and rinse with PBS.
?. [Tissue Fixation ]
Fix brain tissue with 4% PFA  for 2h
on ice
Perform this step under the fume hood. Wear gloves and avoid inhaling.
?. [Tissue Fixation ]
Wash with PBS for 10 min.
?. [Tissue Fixation ]
Repeat Step 4 twice.
?. [Whole Mount Staining ]
Remove PBS and apply 1.5 ml of TTP for each sample in 2ml round-bottom Eppendorf tube. Place in the incubator, rocking, at   for 45 min
37 °C
- Triton and Tween are detergents used to permeabilize the tissue and allow antibody penetration. - About 30 min into the incubation period, preheat Citric acid solution in steamer   to use in the next step.
?. [Whole Mount Staining ]
Apply 1.5 ml of preheated Citric Acid  to each sample and incubate samples in steamer for 30 min
95 °C
CRITICAL STEP: This step replaces bleaching in other protocols, to eliminate blood vessels and tissue autofluorescence.
?. [Whole Mount Staining ]
Apply Blocking solution and incubate, rocking in the cold room for at least 1h   . Meanwhile prepare the proper concentrations of primary antibodies in blocking solution.
4 °C
Concentrations of primary antibodies depend on their quality and specificity. In general, for the embryonic cerebellum,  double the concentration used in cryosections IF staining is a good starting point to test the antibody in wholemount.
?. [Whole Mount Staining ]
Discard Blocking and apply Primary antibody solution. Incubate in the cold room, rocking, overnight
4 °C
Primary antibody can be recycled and reused within 3-4 weeks, stored at   . For larger tissues and better staining, incubation in primary antibody can be extended to 2-3 days.
?. [Whole Mount Staining ]
PBS wash, rocking, for 10min
4 °C
?. [REAGENT SETUP]
PFA: 4% Paraformaldehyde. Can be stored at  to be used within 3-4 weeks.The following solutions can be prepared in large volumes and stored in room temperature for months:1X Phosphate Buffered Saline (PBS)TP1: 0.1% Tween-20 in PBSTP2: 0.4% Triton in PBSTTP: 50% TP1+ 50% TP2 (Final concentrations: 0.05% Tween-20 and 0.2%Triton)0.01 mM Citric Acid Buffer   TEA: 0.1M triethanolamine in water0.01 mM Citric Acid Buffer
4 °C
Paraformaldehyde is toxic and care should be taken especially when weighing out the powder. Use full PPE including a mask, lab coat and gloves.
?. [Whole Mount Staining ]
Repeat Step 13 twice.
?. [Whole Mount Staining ]
Let samples sit on bench for 5-10 min to cool down . Meanwhile, prepare Blocking solution: 10% NDS in TTP , and keep on ice.
0 Room temperature
- Total volume of blocking solution depends on sample size and number of samples. Account for blocking, primary and secondary antibody solutions.for example: ~200 μl per sample, per step, is largely enough for dissected early embryonic cerebellum.
?. [Whole Mount Staining ]
Apply secondary antibodies 1/400 in Blocking solution at  for 3h  (leave overnight for best results and large samples)
4 °C
The samples are photosensitive beyond this step. Keep in the dark or wrap in foil through all the following steps.
?. [Clearing]
Prepare fresh 30%TEA/40%Formamide/30%Water mixture (1.5 ml per sample) and incubate the samples in mixture, rocking, in the cold room for 1h.
4 °C
Prepare solutions containing Formamide under the fume hood. Wear gloves and avoid inhaling.
?. [Whole Mount Staining ]
PBS wash, rocking, for 10 min
4 °C
?. [Whole Mount Staining ]
PBS wash 10min twice. (You can stop at this point and keep the samples in PBS at 4°C overnight, to continues with clearing the next day).
?. [Clearing]
Prepare fresh 60%TEA/25%Formamide/15%Water mixture (1.5 ml per sample) and incubate the samples in mixture, rocking, in the cold room for 4h.
4 °C
Can be extended to overnight for larger tissues.
?. [Clearing]
Prepare fresh 70%TEA/15%Formamide/15%Water mixture (about  1.5 ml per sample) and incubate the samples in mixture, rocking, in the cold room for 2h. Meanwhile, prepare 50 ml of fresh Imaging solution : 40 ml Glycerol + 10 ml TEA.
4 °C
?. [Clearing]
Wash samples in 1.5 ml of (50% imaging solution + 50% Mixture in step 19) a gradual transition to glycerol with gentle mixing, and incubate, rocking, for about 15 min
4 °C
?. [Clearing]
Change to 100% of imaging solution and incubate, rocking, for about 15 min .
4 °C
?. [Clearing]
Store the samples in fresh imaging solution, which is expected to have a refractive index of 1.45 (matching Zeiss lightsheet Z1 X5 lens) and will also be  used to fill the light sheet imaging chamber, and storing the samples
4 °C
Keep the samples in the imaging solution, rocking in the cold room, for at least 24h before imaging (few days for larger samples).
?. [Whole Mount Staining ]
Discard TTP and rinse with PBS.
?. [Whole Mount Staining ]
Discard the Citric acid and rinse with PBS.