|From:||Gayle Callis <firstname.lastname@example.org>|
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 <email@example.com>
>>Subject: Insect processing tips --- long
>>To: "J.L. Turnbull" <firstname.lastname@example.org>
>>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
>>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...
>>"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
>> > sections in paraffin at 56 degrees. I rehydrated by standard
>>> 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
>>> 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.
>>> 1) the protcol said that the iodine step was to remove the 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
>>> 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
>>> 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
>>> 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
>>> 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)
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610
404 994-4303 (FAX)
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