Immunostaining of Tissue Specimens:
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 of interest and put into fixative solution.
3. The time the tissue spends in fixative has an impact on which antibodies will work; many antibodies bind epitopes that are destroyed by fixatives, so we recommend starting with a very mild fixation. Accordingly, we let tissues sit only 1-2 hours in fixative at 4°C and then wash 3 times in PBS with mild shaking. After this procede to 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 1-2 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 pure primaries to ~1 microgram/ml and make up in PBS plus 0.1% goat serum or BSA. 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.