Re: DMSO/IHC

From:Connie McManus <conmac@cc.usu.edu>

Karen,
Thanks for your info on DMSO and antigenicity.  Also, thanks for the
caution in using DMSO with formaldehyde.  I know better, but failed to
pass this along.  

Connie M.



Karen Larison 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)=2E
> >
> >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=2E
> >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=2E
> >>
> >>  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
-- 
ࡱ



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