Jul 29, 2022
Light sheet Sample Processing - Mouse Brain
- 1University of Connecticut
Protocol Citation: Nagham Khouri-Farah, James Li 2022. Light sheet Sample Processing - Mouse Brain. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7ykmv5d/v1
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: June 11, 2022
Last Modified: July 29, 2022
Protocol Integer ID: 64427
Keywords: Clearing , Optical, Brain, light-sheet, Lightsheet, Zeiss, whole mount
Funders Acknowledgement:
NIH_F31
Grant ID: F31NS124264
Abstract
An improved method for light-sheet imaging sample preparation. We use this protocol to process brain tissue (cerebellum) for light sheet imaging. First, we fix the tissue overnight at 4C, then we steam the tissue in Citric acid to retrieve epitopes after crosslinking and bleach the autofluorescence of tissue and blood vessels. This simple step saves time avoiding further bleaching steps in other staining protocols and improves the quality of antibody staining. We adapted pre-staining clearing delipidation using SDS in boric acid to reach optical transparency (McCreedy et al. 2021). For staining, we use conventional immunofluorescence. We proceed with post-staining clearing, based in concept on RTF method (Yu et al. 2018) with modifications. The final imaging solution of 80% glycerol should already have a refractive index of 1.45 matching that of Zeiss lightsheet Z1 X5 lens, and used to store the samples and fill the light-sheet microscope chamber for imaging and for sample storage.
Guidelines
Whole mount staining is based on conventional Immunufluorescence staining method with the added steps of steaming in Citric acid to bleach the samples.
Materials
- Phosphate Buffered Saline (PBS)
- Paraformaldehyde (PFA)
- Triton X-100 (Sigma, 9002-93-1)
- 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)
- Boric Acid
- NaOH
- SDS
- 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.
SDS causes respiratory tract irritation in solid powder form. Avoid contact with skin and eyes. Avoid formation of dust and aerosols.
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)
- 0.01 mM Citric Acid Buffer (6 )
- TEA: 0.1 M Triethanolamine in water
- 10% SDS
- BA Buffer (8.5 ): Dissolve 61.83 g of Boric Acid and 12 g of sodium hydroxide pellets (NaOH) in 900 mL of ddH2O. Stir until fully dissolved and clear. Add ddH2O up to final volume of 1L.
- BBT: 800 mL ddH20, 200 mL BA buffer, and 1 mL of TritonTMX-100.
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
Dissect the mouse brain tissue and try to peel off the meninges. Rinse with PBS.
Note
The cerebellum is our tissue of interest, however, this method can be applied to any other neuronal tissue (forebrain, cerebellum, brainstem, spinal cord,...).
Fix tissue with 4% PFA4 °C Overnight
Safety information
Apply PFA under the fume hood. Wear gloves and avoid inhaling.
Wash with PBS for 10 min.
Repeat Step 4 twice.
Delipidation
Delipidation
45m
45m
Apply 2 ml of preheated Citric Acid to each sample and incubate samples in steamer for 45 min 00:45:00
95 °C
Note
CRITICAL STEP: This step replaces bleaching in other protocols, to eliminate blood vessels and tissue autofluorescence.
45m
Let samples sit on bench for 5-10 min to cool down Room temperature . Meanwhile, prepare 12.5 mL/sample of SDS delipidation solution by combining 10 mL of 10% SDS with 2.5 mL of 1 M boric acid buffer.
Note
Do not store the SDS delipidation solution at RT for more than 1 day since SDS may precipitate in boric acid buffer.
Rinse the samples with PBS.
Transfer tissue from PBS to 12.5 mL of SDS delipidation solution in a 15 mL conical. Seal lid with parafilm and incubate at 37 °C on a nutating rocker.
Replace SDS delipidation solution every other day until the tissue is optically transparent (5-7 days). 120:00:00
Note
Avoid leaving tissues in SDS clearing solution after becoming optically transparent, as this can lead to sample degradation.
5d
Transfer the sample to 14 mL of BBT and rotate at Room temperature for 00:30:00 min on tube revolver to wash out the residual SDS.
30m
Repeat step 11 twice
Leave the sample in BBT solution Overnight rotating at Room temperature
45m
Repeat steps 11-13
Staining
Staining
2d 0h 45m
2d 0h 45m
Prepare the staining buffer: primary antibody + 1ml BBT + 2% (v/v) normal donkey serum (NDS)
Note
Concentration of primary antibody varies.
Transfer the tissue into the staining buffer with primary antibodies in 2 mL tube. Protect from light and rotate for 2 days at RT on tube revolver.48:00:00 Room temperature
2d
Wash tissue with BBT solution with 0.01% with 3 buffer changes within 1-2 h on a tube revolver at RT.
Prepare secondary antibodies 1/500 in 1ml BBT + 2% (v/v) normal donkey serum (NDS).Overnight Room temperature
Note
Filter staining buffer with secondary antibodies using syringe filter to remove any aggregates of secondary antibody that may affect IF labeling.
45m
Wash tissue with BBT solution with 0.01% with 3 buffer changes within 1-2 h on a tube revolver at RT.
Post-staining clearing
Post-staining clearing
1h 45m
1h 45m
Prepare fresh 50%TEA/30%Formamide/20%Water mixture (12.5 ml per sample) and incubate the samples in mixture, rocking. Room temperature Overnight
Prepare fresh 70%TEA/15%Formamide/15%Water mixture (about 1.5 ml per sample) and incubate the samples in mixture, rocking 01:00:00
1h
Meanwhile, prepare 50 ml of fresh Imaging solution : 40 ml Glycerol + 10 ml ddH2O.
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 01:00:00
1h
Change to 100% of imaging solution and incubate, rocking, for about 15 min
Store the samples at 4 °C in fresh imaging solution
Note
Imaging solution (80% Glycerol) is expected to have a refractive index of 1.45 (matching Zeiss lightsheet Z.1 X5 lens) and will also be used to fill the light sheet imaging chamber.