X-Message-Number: 24496
From: "Basie" <>
Subject: Murphy's law 
Date: Wed, 11 Aug 2004 01:11:44 -0400

Who do you think has the best chance. If one assumes that chemical damage
due to freezing or cross linking can be fixed some time in the future then
fixed and then frozen gives one the best chance because given Murphy's law a
melt down will occur. No one will know that a melt down occured until a
hundred years hence. So why take a chance.

Basie

Immunostaining of Tissue Specimen:

    Here are two alternative procedures, both of which work well. The more
well established method is to perfuse the animal with fixative while under
anesthetic. This requires some surgical skills, since the perfusion has to
done in the aorta, which can only be reached by opening the chest cavity.
Obviously the animal has to be fully anesthetized, and is in most cases a
small rodent such as a rat. You will also need to obtain approval from your
local animal care authorities to do this. It is a lot easier to sacrifice
the animal first, dissect out the tissues you want and just drop them into
fixative. This works fine, and can be performed on animals killed at
slaughterhouses or for other purposes.
    Fixed tissues are then impregnated with sucrose, which acts as a
cryoprotectant, preventing ice crystal formation during freezing. Specimens
are then rapidly frozen and sectioned on a cryostat.

A. Perfusion.

1. Perfuse animal with heparinized normal saline after anesthetizing with
pentabarbitol.

2. When blood has been washed out, perfuse in 4% paraformaldehyde fixative
freshly made up in PBS (best), 4% paraformaldehyde in PBS not so fresh
(O.K.) or 1+9 solution of Fisher Formalin in PBS (also O.K. for most
purposes). Dissect out tissues and proceed as in C below.

B. No Perfusion.

1. Dissect out tissues and drop into fixative at room temperature. Fixative
is 4% paraformaldehyde fixative freshly made up in PBS (best), 4%
paraformaldehyde in PBS not so fresh (O.K.) or 1+9 solution of Fisher
Formalin in PBS (also O.K. for most purposes). To aid in the penetration of
the fixative cut tissues down to steaks of no more than 0.5 cm thickness.
Also its good to gently shake specimens on a rocker platform to allow
fixative to penetrate tissues. For pilot experiments with antibodies you
have n't tried before 1 hour fixation is enough. Can leave longer or
overnight at 4 C if you know the particular antigen/antibody reaction can
stand this.

2. Move specimens to PBS and proceed to C below.

C. Sucrose impregnation and rapid freezing.

1. Transfer the tissue to 20% sucrose in PBS, leave overnight at 4 C.

2. Transfer the tissue to 30% sucrose in PBS, leave at 4 C to impregnate
fully. Typically when the tissue sinks, it is fully impregnated, which
usually take 2-3 days.

3. Rapidly freeze in isopentane (a.k.a. 2-methylbutane) cooled with liquid
nitrogen. To do this put 5-10 mls of isopentane in a small polystyrene or
other plastic container; a 35mm film container or a 50ml beaker is fine for
this purpose. Then put that in a larger plastic container, such as a 500ml
polystyrene or polypropylene beaker in which you put ~100 ml liquid
nitrogen. Let the liquid nitrogen cool the isopentane til you can see it
start to freeze, which starts at -160 C. The isopentane freezes from the
bottom of the container, forming round white pearl like objects. When this
happens drop the specimen directly into the bottom of the container. Take
specimen out using plastic or plastic coated forceps (they tend to stick to
metal forceps) and either store them at -70 C or proceed immediately to
sectioning. Tissue should now not be allowed to thaw out until after
sectioning.

4. Mount on cryostat and cut 5 to 20mm thick sections. Cut at -25 C and
mount sections on "+" marked slides or subbed slides. Store at -20 C
or -70 C or proceed to immunostaining right away.

D. Immunostaining with fluorescent secondary antibodies

1. Optional; Wash sections for 30 seconds in cold Acetone (-20 C). This step
allows better antibody penetration, but may wash out your antigen,
especially in the case of cytosolic proteins. Let section dry before next
step.

2. Optional; block non-specific binding by incubation of section with 1%
goat serum in PBS for 1 hour at 37 C.

3. Draw circle round dry section with PAP pen to prevent loss of antibody.
Add primary antibodies; Typically dilute primaries to 1mg/ml and make up in
PBS plus 0.1% goat serum. Can apply rabbit and mouse antibodies at same time
for double label immunocytochemistry. Typically incubate for 1 hour at 37 C
or 2 hours at room temperature or overnight at 4 C.

4. Wash sections at least 3 times, at least 5 minutes each time, in PBS. To
reduce background can include 0.1% Tween 20 in PBS.

5. Apply secondary antibodies. Best are goat anti-mouse and goat anti-rabbit
antibodies from Molecular Probes, and best fluorochromes are the ALEXA
conjugates; ALEXA 488 and ALEXA 594. The 488 is very similar in spectral
properties to FITC, and the 594 is similar to rhodamine and Texas red. Both
are resistant to bleaching and work at 1 microgram/ml dilution, which is
1:2,000 from the 2 milligram/ml material obtained from Molecular Probes.
Incubate with secondaries in PBS plus 0.1% goat serum. Typically incubate
for 1hr at 37 C or 2 hours at room temperature or overnight at 4 C.

6. Wash sections at least 3 times, at least 5 minutes each time, in PBS. To
reduce background can include 0.1% Tween 20 in PBS.

7. Mount in mounting medium, a useful one being Vectasheild mounting medium
with DAPI, which allows you to stain nuclei with the DNA intercalating
fluorescent dye DAPI, which you can see on a fluorescence microscope fitted
with appropriate blue filters.

8. View on fluorescence microscope.

 EnCor Biotechnology Inc. 2004

Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=24496