TITLE: Light sheet Sample Processing - Mouse Brain
AUTHORS: Nagham   Khouri-Farah, James  Li

[DESCRIPTION]
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. 
Tissue clearing process is based on PACT (McCreedy et al. 2021) and RTF (Yu et al. 2018) methods.

[STEPS]
SECTION: REAGENT SETUP
1. 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 (pH 6)
TEA: 0.1 M  Triethanolamine in water
10% SDS
BA Buffer (pH 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.
 


SECTION: Tissue Fixation
2. Dissect the mouse brain tissue and try to peel off the meninges. Rinse with PBS. 

 



SECTION: Tissue Fixation
3. Fix tissue with 4% PFA4 °C   
 



SECTION: Tissue Fixation
4. Wash with PBS for 10 min.
SECTION: Tissue Fixation
5. Repeat Step 4 twice.
SECTION: Delipidation
6. Apply 2 ml of preheated Citric Acid  to each sample and incubate samples in steamer for 45 min 45 min 
95 °C 

 

SECTION: Delipidation
7. 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.
 


SECTION: Delipidation
8. Rinse the samples with PBS.
SECTION: Delipidation
9. 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.

SECTION: Delipidation
10. Replace SDS delipidation solution every other day until the tissue is optically transparent (5-7 days). 7200 min 
 


SECTION: Delipidation
11. Transfer the sample to 14 mL of BBT and rotate at Room temperature for 30 min min on tube revolver to wash out the residual SDS.

SECTION: Delipidation
12. Repeat step 11 twice
SECTION: Delipidation
13. Leave the sample in BBT solution 45 min rotating at Room temperature

SECTION: Delipidation
14. Repeat steps 11-13
SECTION: Staining
15. Prepare the staining buffer: primary antibody + 1ml BBT + 2% (v/v) normal donkey serum (NDS)
 

SECTION: Staining
16. 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.2880 min Room temperature 

SECTION: Staining
17. Wash tissue with BBT solution with 0.01% with 3 buffer changes within 1-2 h on a tube revolver at RT. 
SECTION: Staining
18. Prepare secondary antibodies 1/500 in 1ml BBT + 2% (v/v) normal donkey serum (NDS).45 min Room temperature
 



SECTION: Staining
19. Wash tissue with BBT solution with 0.01% with 3 buffer changes within 1-2 h on a tube revolver at RT. 
SECTION: Post-staining clearing
20. Prepare fresh 50%TEA/30%Formamide/20%Water mixture (12.5 ml per sample) and incubate the samples in mixture, rocking. Room temperature 45 min 

SECTION: Post-staining clearing
21. Prepare fresh 70%TEA/15%Formamide/15%Water mixture (about  1.5 ml per sample) and incubate the samples in mixture, rocking 60 min 

SECTION: Post-staining clearing
22. Meanwhile, prepare 50 ml of fresh Imaging solution : 40 ml Glycerol + 10 ml ddH2O.
SECTION: Post-staining clearing
23. 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 60 min 

SECTION: Post-staining clearing
24. Change to 100% of imaging solution and incubate, rocking, for about 15 min 
25. Store the samples at 4 °C in fresh imaging solution