Re: DMSO/IHC

From:Gayle Callis <uvsgc@montana.edu>

What species?  Mouse embryos, zebra fish???


At 02:43 PM 4/19/01 -0800, you wrote:
>Connie and Jenny,
>
>We routinely use 1% DMSO in our wash/dilution buffers when we do IHC.
>We do a lot of IHC on whole mount embryos and the DMSO is required
>for adequate penetration of the reagents.  I've also tested various
>concentrations of DMSO.  1% DMSO works great, but greater
>concentrations appear to destroy antibodies, etc.  In addition, we
>have used 1% DMSO in our fixative with no deleterious effect on
>antigenicity.  And we have used 10% DMSO to pretreat our fixed
>embryos.  This is required to permeabilize the embryo if denaturing
>fixatives have been used.  Antigenicity remains unaffected if the
>specimen is washed thoroughly.
>
>Use caution whenever you add DMSO to noxious chemicals like
>fixatives.  DMSO is a carrier molecule and reportedly transports
>molecules such as DAB and formalin through skin and gloves.  Wear
>gloves, but also change them immediately if you have spills.
>
>Karen in Oregon
>
>
>
>
>
>
>>Date: Thu, 19 Apr 2001 14:38:32 -0600
>>From: Connie McManus <conmac@cc.usu.edu>
>>Subject: Insect processing tips --- long
>>To: "J.L. Turnbull" <jlt25@hermes.cam.ac.uk>
>>Cc: histonet@pathology.swmed.edu
>>
>>Jenny,
>>
>>I have worked with insects before and would like to pass along some tips
>>for working with them. We always used Bouins fixative for light
>>microspcopy and the glutaraldehyde buffered in a Phosphate buffer for
>>EM. Because you're doing IHC, I don't think the Bouin's would be a good
>>choice of fixative.  A word on cacodylate buffer:   My husband's lab
>>(the campus EM facility) has ceased using cacodylate buffer altogether
>>because of it's toxicity.  It is also probably more of a "fixative"
>>(notice the quotes) rather than a buffer.  It probably just simply
>>poisons the cells, rather than form linkages with proteins.  My husband
>>is the real expert on this, we just have these discussions over dinner
>>in our favorite restaurant where he conveys this info along to me. you
>>should see some of the looks we get!  *g*  I digress.
>>
>>Because you're doing IHC on paraffin embedded tissues, I would use 10%
>>Buffered Neutral Formalin with DMSO added to fix tissues in.  The reason
>>to put DMSO in the fixative is discussed below.  This has been a
>>critical ingredient in fixation of insects when I was working with them.
>>HOWEVER, I don't know how DMSO affects IHC.  If it turns out to be bad,
>>don't add it to the BNF formula.  Which case, I can't guarentee  how
>>well fixed your caterpillars will be.  Anyway, since you're a novice to
>>histology, I'll provide the formulation for this fixative (aren't I a
>>sweet heart?  *g*)
>>
>>37% formaldehyde ---- 100 mL
>>DMSO ----------------   20 mL
>>Na2HPO4 -------------  6 g
>>NaH2PO4 -------------  4.5 g
>>DI water ------------ 880 mL
>>
>>dissolve the phosphates in the DI water with the aid of stirring and
>>gentle heat (do not boil or let get so hot you cannot touch the flask).
>>
>>Add the formaldehyde and DMSO in a fume hood, wearing goggles and
>>nitrile gloves.  Mix well.  Store this at room temperature in a tightly
>>sealed, unbreakable container.  Do not use if a white precipitate forms
>>or if it has been frozen.  Potassium phosphates may be used instead of
>>the sodium.  Some people I have worked with also add 2 -5% glacial
>>acetic acid, but i don't know how that will affect the IHC.  I would
>>avoid using acetic acid at this time.
>>
>>DMSO is added to the fixative because all insects have a tough,
>>impermeable outer layer, the integument, that is designed to prevent
>>environmental things from entering the insect.  This includes larvae
>>(caterpillars are larvae).  If you're processing the entire caterpillar,
>>you will need to make slits in the sides with a very sharp razor blade
>>or scalpel blade.  These should be just deep enough to allow fixatives
>>and processing solutions to get inside.  The DMSO will help with the
>>infiltration.  Vacuum pressure is also very important to use in insect
>>histology to remove air bubbles trapped inside the body.  However, do
>>not use vacuum for long periods of time, just enough to remove the air
>>and let the solutions inside the body, then return to normal atmosphere.
>>
>>Although I have never used microwave processing, my husband and others
>>tell me it's the greatest thing since hotcakes. If you have access to a
>>microwave, there are many people here who can tell you better how to use
>>it.
>>
>>I hope this makes some sense... I've been running around doing things in
>>the lab, then coming back to this post, so it might sseem disconnected.
>>hope it helps at any rate...
>>
>>cheers!
>>
>>Connie McManus
>>
>>
>>"J.L. Turnbull" wrote:
>>>
>>>  I am a biochemist by trade, but ahve wentured into some histology, rather
>>>  unsuccessfully.  None of my biochemist contacts know anything
>>>about about this
>>>  so i'd really appreaciate any assistance.
>>>
>>>  I have been trying to locate protein insecticides in sections of
>>>  caterpillar gut with a  peroxidase-conjugated antibody.  I've done quite
>>>  a few runs and changed many variables - still brown.
>>>
>>>  I based my protcol on a published paper using similar insects.
>>>  I fixed in 2.5% gluteraldehyde 0.5M cacodylate for 24 hours.  Anatomy
made
>>  > sections in paraffin at 56 degrees.  I rehydrated by standard
techniquwes
>>>  and continued roughly as follows:
>>>
>>>  Incubate in iodine/K iodide
>>>
>>>  Equilibrate in Tris, Saline, Triton buffer.
>>>
>>>  Antigen revival in 1 mg/ml trypsin 10 min.
>>>
>>>  Quench endogenous peroxidase with 0.03% H2O2 in methanol 20 min.
>>>
>>>  primary monoclonal antibody in either milk. BSA, Haemoglobin or
pre-immune
>>>  serum.  Washed many times.
>>>
>>>  Either peroxidase-conjugated secondary antibody or VECTASTAIN ABC
>>>  avidin-biotin kit.
>>>
>>>  Developed in either DAB or chloronaphthol in methanol,
>>>
>>>  Everything was brown, even antigen-free and antiserum-free controls.
>>>
>>>  Questions:
>>>  1) the protcol said that the iodine step was to remove the sublimate,
what
>>>  sublimate?
>>>
>>>  2) How does the quenching step work?  I used the same recipies plus
>>>  chromogen to develop and it didn't quench that?  Is it just oxidative
>>>  damage?
>>>
>>>  3)  I guessed that endogenous peroxidase was a problem (so haem. was bad
>>>  choice then i realise).  I tried using sodium azide as an inhibitor
(is it
>>>  reversible or not?)  this didn't work either.
>>>
>>>  4) i didn't know i had to filter the DAB - could this be the explanation?
>>>  However. chloronapthol didn't work either.
>>>
>>>  5) the other protocol used Hollandes fixative - would this make a
>>>  difference.
>>>
>>>  6) I tried doing this with whole guts (whilst i made more slides) just to
>>>  see if the sectioning process had done something.  Most went brown but
the
>>>  2 that i hadn't fixed gave a white control and brown sample.  Did i use
>>>  the wrong fixative therefore.  I'm looking mainly at membranes, so i
>>>  suppose methanol would have been better (I'm only just learning this
>>>  histology business)
>>>
>>>  Please help (and sorry it's not more concise)
>>>
>>>  Jenny
>>
>>--
>>ࡱ
>
>
>
>
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)




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