Light sheet Sample Processing - Mouse Brain
Nagham Khouri-Farah, James Li
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.
Steps
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
Dissect the mouse brain tissue and try to peel off the meninges. Rinse with PBS.
Fix tissue with 4% PFA4°C
Wash with PBS for 10 min.
Repeat Step 4 twice.
Delipidation
Apply 2 ml of preheated Citric Acid to each sample and incubate samples in steamer for 45 min 0h 45m 0s
95°C
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.
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°Con a nutating rocker.
Replace SDS delipidation solution every other day until the tissue is optically transparent (5-7 days). 120h 0m 0s
Transfer the sample to 14 mL of BBT and rotate at Room temperature for 0h 30m 0s min on tube revolver to wash out the residual SDS.
Repeat step 11 twice
Leave the sample in BBT solution 0h 45m 0s rotating at Room temperature
Repeat steps 11-13
Staining
Prepare the staining buffer: primary antibody + 1ml BBT + 2% (v/v) normal donkey serum (NDS)
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.48h 0m 0s Room temperature
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).0h 45m 0s Room temperature
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
Prepare fresh 50%TEA/30%Formamide/20%Water mixture (12.5 ml per sample) and incubate the samples in mixture, rocking. Room temperature 0h 45m 0s
Prepare fresh 70%TEA/15%Formamide/15%Water mixture (about 1.5 ml per sample) and incubate the samples in mixture, rocking 1h 0m 0s
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 1h 0m 0s
Change to 100% of imaging solution and incubate, rocking, for about 15 min
Store the samples at 4°C in fresh imaging solution
