The University of Pennsylvania Health System
 

 

 

 

Clearing Agents:  Click on a heading to jump to that section.

 

 

Glycerol     Methyl Salicylate     BABB

 

 

 


1.  Buffered Glycerol

To make 50ml: Mix 25 ml glycerol, 5 ml 10X PBS, pH 7.4, 20 ml dH2O. Immerse cells or tissue in three changes to ensure penetration. Mount with fresh buffered glycerol, coverslip and seal with nail polish.

Refractive index = 1.42


 

 

 

 


2.  Methyl Salicylate

Cell Protocol:

Use without buffering. Immerse cells in 3 - 4 changes to eliminate residual H2O. Adjust time to insure adequate water removal. Mount with fresh methyl salicylate and seal coverslip with nail polish.


Tissue Protocol:

(This procedure has been worked out for clearing thick slices of rat brain tissue (~0.5 mm thick) previously fixed with 4% paraformaldehyde.)

1. Wash 2x in fresh PBS, 5 min each.

2. Wash 2x in dH2O, 5 min each.

3. Dehydrate with a graded ethanol series: (20%, 30%, 50%, 70%, 95%), 5 min each

4. Finish dehydration with two100% ethanol washes, 5 min each.

5. Begin clearing by soaking in methyl salicylate, 30 min

6. Change for fresh methyl salicylate, soak until clear.

6. Mount sample in fresh methyl salicylate, coverslip, seal with nail polish.

 

Refractive index = 1.54


 

 


3.  BABB (benzyl alcohol - benzyl benzoate)

BABB: benzyl alcohol (Sigma B-1042)/ benzyl benzoate (Sigma B-6630) 1:2 ratio

1.       Dehydrate with graded MeOH series before sinking tissue into BABB as follows:

a.       25% MeOH (25 % MeOH / 75 % PBT (PBS with 0.1% Tween-20) for 5 min

b.       50% MeOH,  5 min,

c.       75% MeOH,  5 min,

d.       100% MeOH,  2x 5 min each

e.        50% MeOH - 50% BABB, 2x 5 min each

 2.       “Sink” tissue into BABB solution ON at RT.

3.      Mount in barest minimum droplet of BABB, coverslip, seal with nail polish.

 

Refractive index = 1.51



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