Hey Folks --
More thoughts:
>In Ian's example, unless I read it wrong, he was describing solvent
>treatment on sections. He did not describe, the fixation itself which
>I think important to the success of his approach and it seems to be
>in the context of TEM more than confocal, which usually doesn't
>include 50-80 um sections but 0.5 um sections at best. Ian, perhaps
>you could clarify? In any event, detergents work by disrupting
>hydrophobic structures, which means that they can kill antibody
>probes as well as epitopes of interest. So in that I agree, fully.
>Keep detergents out of the antibodies. Besides, once detergent has
>been used on the sample, the permeabilizing is done. Wash it away,
>the holes will stay.
My understanding is that for saponin permeabilization, the membrane holes
will "reseal" unless you keep the detergent in all of the subsiquent steps.
I prefer saponin (0.1%) for most of the thick section work I've done so
far. It is not as harsh on the morphology (as evaluated by DIC) as Triton,
and gets antibodies in deeper than Tween. I've not found that it hurts the
antibodies I've used.
>4. Yes, freeze thaw cycles are bad, but it will really depend on the
>antibody as to how tolerant it is and as to what the other proteins
>it is stored with, if any. The issue is denaturation and loss of
>specificity. Inclusion of other passive proteins such as BSA to 10
>mgs/ml can help. Our labs practice has been to upon arrival of
>antibody from Jackson, Calbio, etc., to aliquot 2 ul into 0.5-1.5 ml
>plastic vials and store at -80 deg C. Use a vial for a few days after
>diluting with incubation media, which usually involves Pierce
>SuperBlock or equivalent and store between uses at 4 deg C. After a
>few days, throw it out. One freeze, one thaw. Why even make it an
>issue except for antibodies that don't like being put at -80 deg.
>There are some. The glycerol approach seems to work for them stored
>at -20 deg. C.
>
I've used Jackson Immuno (no relation to The Jackson Laboratory)
antibodies for many years and have never had any problem just rehydrating
with water and freezing at -20C in 50 ul aliquots. One aliquot is kept at
4C and used as a stock for experiments, leftover diluted antibody is
discarded. Again, only one freeze/thaw. I've used concentrated stock that
has been sitting at 4C for over a year with excellent results. That being
said, I do like the idea of flash freezing small aliquots and storing at
-80C so that you really know that you are using equivalent reagents for all
of your experiments, but practically it's not feasable right now (-80C
space is very limited and very far away).
>
>>Hi All - Ian's last reply was very interesting, as I've been wondering about
>>detergent alternatives to permeabilisation for antibodies some time. Any
>>opinions among people about comparative merits of Triton, Tween, saponin, on
>>morphology if you have to go the detergent route. Also what alternatives
are
>>there - freeze-thaw? for thick specimens (say 50 -80 microns
>>vibratome cut brain
>>sections). Ian, could you give a reference or detailed protocol for the
DMSO
>>method? How uniform is the labelling through the thickness of the section?
>>regards,
>>
>>Martyn Evans
>>SmithKline Beecham
>>Harlow UK
>
>--
>
>Mario M. Moronne, Ph.D.
>NanoMed Technologies
>ph (510) 528-2400
>FAX (510) 528-8076
>Berkeley, CA
>94706
>
Cheers, Greg.
Greg Martin
Light/Confocal Microscopy
Biological Imaging Service
The Jackson Laboratory
600 Main Street
Bar Harbor ME 04609
207-288-6310
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