Leaf clearing techniques?

mel turner mturner at acpub.duke.edu
Thu Jul 28 23:52:05 EST 1994


In article <30jr6c$4qa at mustang.alleg.edu> barosb at alleg.edu (Biomancer) writes:

>I'm doing research with epidermal and mesophyll cells, especially  
>counting cells per unit area after ABA treatment in Hippuris  
>vulgaris...does anybody have a good, quick clearing method that will   
>remove everything excpet cell walls?  I've  been using 90% EtOH followed  
>by 7% NaOH, but it doesn't clear enough that I can see mesophyll cell  
>walls.  Any suggestions?  Most of the readings that I've looked at are  
>for the staining of xylem, but I don't need to see the xylem, and some  
>even degrade cellulose, which for me would be bad.

Try heating or even autoclaving  specimens in lactophenol  --   autoclaving 
often works well, giving cleared specimens  in a few minutes.    First try 
looking at them unstained, in lactophenol  (maybe use phase contrast 
microscopy or Nomarski).  To stain you will probably have to wash out the 
clearing medium,  stain them  & dehrydrate with alcohols and move them into a 
high refractive index medium  like xylene or methyl salicylate  (or through 
xylene into a permanent slide- mounting resin).   Stains you might try include 
tannic acid w/ ferric chloride, and fast green  ( maybe try a red or orange 
filter to observe /photograph the latter).  Cleared specimens only become very 
glassy/ transparent in the later (xylene, etc.) steps.

If lactophenol doesnt do it , try following up the NaOH step with a little 
very dilute bleach  (Chlorox, ca 10%)  just until they are white  (too long 
and they will macerate completely), before staining, dehydrating etc.

Good luck, mdt



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