Worm Breeder's Gazette 14(5): 23 (February 1, 1997)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Another fixation method for immunohistochemistry using whole worms

Michael L. Nonet

Dept. of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110 (nonetm@thalamus.wustl.edu)

I have developed a fixation protocol which provides an alternative set
of conditions to test antisera that work poorly on paraformaldehyde
fixed worms (e.g. the Finney protocol).  A Bowin's fixative of 75 ml
saturated picric acid [real nasty stuff- explosive in crystal form!], 25
ml of formalin, and 5 ml of glacial acetic acid is made. Many fly labs
use this fixation solution which may be stored at 4=B0C for many months. 
Nematodes grown on NGM plates are washed off the plate using 2 to 3 ml
of H2O and spun down in a glass 12 ml conical tube in a clinical
centrifuge at room temp.  The supernatant, save 50 ul, is removed by
aspiration.   A mix of 400 ul of Bowin's, 400 ulmethanol and 10 ul of
BME is placed in the glass tube to initiate fixation and subsequently
the worms are transferred to an eppendorf tube using a glass pipette
(600:200:10 Bowin's: MeOH: BME also works).  The tube is rocked on a
Labquake shaker at RT for 30 minutes, then quick frozen in liquid N2. 
The tube is quickly thawed under running hot water until the solution
melts (but before it warms up past RT).  The tube is rocked an
additional 30 minutes.  A solution of  BTB [ 1X Borate buffer,
0.5% Triton X-100, 2%  BME] is made from 50X borate buffer ( 1.0 M
H3BO3, 0.5 M NaOH).  The worms are spun from fixative in a centrifuge [
2-3 seconds in an eppendorf 5415C ].  The fixative is aspirated, and 1.4
ml of wash solution is added, rocked a few seconds to mix.  Spinning and
aspiration completes the first wash.  Two more washes are done, and the
worms are then resuspended again in 1.0 ml BTB ( in the lower volume
worms move more during rocking).  The worms still have a yellow tinge
due to the picric acid at this point.  The worms are now rocked for
approximately 1 hr at RT.  The speed at which the yellow picric acid
destains is a good sign of how well the worms are going to stain.  If
the worms remain yellow after an hr of washing, they usually end up
impermeable to antibodies.  Wash again with BTB and incubate an
additional 2 or 3 hrs.  Wash with BT (BTB lacking BME) one time, and AbA
(1X PBS, 1.0% BSA, 0.5% Triton X-100, 10 mM NaAzide) solution two times. 
Incubate 30 minutes or more then begin standard Ab incubations.  I never
switch tubes during this procedure.  I store these worms at 4 C and they
often stain relatively effectively a month later (and sometimes they
        In my hands this procedure gives good staining in greater than
90% of the worms.  Our antibodies to RAB-3 and SNB-1(synaptobrevin/VAMP)
work much better on Bowin-fixed worms than on well permeabilized 'Finney
protocol'-fixed worms.  UNC-64 (syntaxin) Ab works equally well in the
protocols, and SNT-1 (synaptotagmin) Ab works much better using the
Finney fixation than the Bowin's fixation, although staining is still
reasonable in Bowin's.  Thus, Bowin's fixation provides an alternative
condition that may reveal different epitopes on proteins.  I have not
altered fixation length very much, but I have found that increased
fixation does reduce permeability.  A 1 hr fixation seems to be a good
compromise between fixation length and retaining permeability, but I
would suggest reducing fixation to 30 minutes if you find your worms are
not permeable when you try the procedure.  I am relatively certain that
small changes in the temperature during fixation can cause dramatic
changes in the extent of fixation.