Aug 04, 2020
LightsheetSampleProcessing-EmbryonicBrain
- 1University of Connecticut
- Optical Clearing of Tissue
- Li lab
Protocol Citation: Nagham Khouri-Farah 2020. LightsheetSampleProcessing-EmbryonicBrain. protocols.io https://dx.doi.org/10.17504/protocols.io.bi8jkhun
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 01, 2020
Last Modified: August 04, 2020
Protocol Integer ID: 39915
Keywords: Lightsheet, Imaging, Brain, Embryo, Clearing, Wholemount, Whole mount, Light sheet,
Abstract
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. An added ingenious step 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 H2O2 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.
Guidelines
Whole mount staining is based on cryosection Immunufluorescence staining method with the added steps of Triton/Tween permeabilisation and steaming in Citric acid to bleach the samples.
Materials
MATERIALS
Triethanolamine hydrochlorideSigma – AldrichCatalog #T1502
- Phosphate Buffered Saline (PBS)
- Paraformaldehyde (PFA)
- Triton X-100 (Sigma, 9002-93-1)
- Tween-20 (Sigma, P5927)
- Citric Acid Anhydrous (Fisher Scientific, BP339)
- NDS: Heat-inactivated Donky Serum stored at -20 C
- Primary antibodies to epitope of interest. (up to three from different species)
- Species-specific fluorescent secondary antibodies. (non-overlapping excitation/emission ranges)
- Triethanolamine hydrochloride 99.5% (Sigma, T1502)
- Formamide 99.0% (Sigma, F7503)
- Glycerol (Fisher Scientific, BP229-1)
- 2 ml round-bottom Eppindorf tubes
- Oster food steamer
Safety warnings
Paraformaldehyde is toxic and care should be taken especially when weighing out the powder. Use full PPE including a mask, lab coat and gloves.
Formamide is toxic. prepare solutions under the fume hood.
REAGENT SETUP
REAGENT SETUP
PFA: 4% Paraformaldehyde. Can be stored at 4 °C 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 PBS
- TP2: 0.4% Triton in PBS
- TTP: 50% TP1+ 50% TP2 (Final concentrations: 0.05% Tween-20 and 0.2%Triton)
- 0.01 mM Citric Acid Buffer 6.0
- TEA: 0.1M triethanolamine in water
Safety information
Paraformaldehyde is toxic and care should be taken especially when weighing out the powder. Use full PPE including a mask, lab coat and gloves.
Tissue Fixation
Tissue Fixation
2h 40m
2h 40m
Dissect the embryonic brain tissue and rinse with PBS.
10m
Fix brain tissue with 4% PFA for 2h 02:00:00 On ice
Safety information
Perform this step under the fume hood. Wear gloves and avoid inhaling.
2h
Wash with PBS for 10 min.
10m
Repeat Step 4 twice.
20m
Whole Mount Staining
Whole Mount Staining
1d 3h
1d 3h
Remove PBS and apply 1.5 ml of TTP for each sample in 2ml round-bottom Eppendorf tube. Place in the incubator, rocking, at 37 °C for 45 min 00:45:00
Note
- 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 95 °C to use in the next step.
45m
Discard TTP and rinse with PBS.
Apply 1.5 ml of preheated Citric Acid to each sample and incubate samples in steamer for 30 min 00:30:00 95 °C
Note
CRITICAL STEP: This step replaces bleaching in other protocols, to eliminate blood vessels and tissue autofluorescence.
30m
Let samples sit on bench for 5-10 min to cool downRoom temperature . Meanwhile, prepare Blocking solution: 10% NDS in TTP , and keep on ice.
Note
- 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.
5m
Discard the Citric acid and rinse with PBS.
Apply Blocking solution and incubate, rocking in the cold room for at least 1h 4 °C . Meanwhile prepare the proper concentrations of primary antibodies in blocking solution.
Note
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.
1h
Discard Blocking and apply Primary antibody solution. Incubate in the cold room, rocking, overnight Overnight 4 °C
Note
Primary antibody can be recycled and reused within 3-4 weeks, stored at 4 °C .
For larger tissues and better staining, incubation in primary antibody can be extended to 2-3 days.
12h
PBS wash, rocking, for 10min 4 °C
10m
Repeat Step 13 twice.
20m
Apply secondary antibodies 1/400 in Blocking solution at 4 °C for 3h 03:00:00 (leave overnight for best results and large samples)
Safety information
The samples are photosensitive beyond this step. Keep in the dark or wrap in foil through all the following steps.
3h
PBS wash, rocking, for 10 min 4 °C
10m
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).
20m
Clearing
Clearing
7h 30m
7h 30m
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.
01:00:00 4 °C
Safety information
Prepare solutions containing Formamide under the fume hood. Wear gloves and avoid inhaling.
1h
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. 04:00:00 4 °C
Note
Can be extended to overnight for larger tissues.
4h
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. 02:00:00 4 °C
Meanwhile, prepare 50 ml of fresh Imaging solution : 40 ml Glycerol + 10 ml TEA.
2h
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
15m
Change to 100% of imaging solution and incubate, rocking, for about 15 min .4 °C
15m
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
Note
Keep the samples in the imaging solution, rocking in the cold room, for at least 24h before imaging (few days for larger samples).