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Immunohystochemistry protocol for whole mount tissue This protocol is to be carried out at 4oC. Important: keep antibodies in the dark and cold. Stock solutions are to be kept at -80oC. Once an aliquot is thawed it should NOT be frozen again (freezing and thawing peptides and proteins damages them more than keeping them at 4oC) 1) Dissect out the tissue you want to study 2) Fix in 4% paraformaldehide 6 hours to overnight 3) Rinse 6 times over a period of 6 hours in a solution of phosphate buffer + 0.3% TritonX (P-TriX). The detergent Triton X is important to permeabilize the membranes and allow the antibodies to penetrate the cells and bind to intracellular epitopes. 4) Incubate in the primary antibody diluted in phosphate buffer + 0.3% TritonX for 12 or more hours. Add to this 10% serum to reduce non-specific binding of the antibody. The serum should be of the same species as the animal species the secondary antibody (see step 6) was made in. Example: goat serum, if the secondary antibody is a goat anti-mouse antibody. The primary antibody has to be diluted in P-TriX according to specifications of the producer or whoever gave you the antibody. A good rule of thumb to begin with is a dilution of 1:100. If no staining or very faint staining is observed, a lower dilution (1:20 or 1:50) should be tried. If too bright, obviously a higher dilution is necessary. Remember that these antibodies are precious, either because they are expensive or very hard to get. So, calculate and recalculate your dilutions carefully. If the antibody is really hard to get (polyclonal antibodies are always in limited supply; monoclonal antibodies tend to be easier to get and cheaper). If the antibody is supposed to stain an extracellular epitope, the detergent is not necessary. 5) Repeat step 3 (6 rinses for an hour each) 6) Incubate in fluorophore-conjugated secondary antibody diluted to the appropriate concentration (according to manufacturer's specifications or your own experience) in phosphate buffer + 0.3% TritonX for 12 or more hours. No serum is necessary at this step. It is very important to carry out this step in the dark to prevent photobleaching of the fluorophore. 7) Repeat step 3 (6 rinses for an hour each in phosphate buffer without Triton X). Too short washes and the bacjground will be high! 8) Mount the tissue between glass microscope slides. There are 2 good mounting
protocols: The mounting solution is pure methyl salycylate. Sigma's is fine, but there are other sources that seem to do just as well. Be careful with this stuff though. It is not really too toxic (it is just spearmint after all), but you can build up an allergy to it. Many common foods have spearmint in them, which you will then have to avoid. Use both gloves and a fume hood, if possible. In addition to thickness, another determining factor with crustacean ganglia is the permeability of the sheath. If you desheath, 5 minutes should be long enough, but I would say 8 to 10 to be safe. At least 8 or so minutes if you do not desheath. If you want to try 5 minutes, the symptoms of too-short washes are roughly the same as for increasing the alcohol concentration too rapidly: the cells look like they are wrinkled and puckered. Also, since salycylates are not miscible with water, sometimes you will get a milky appearance in the ganglia-basically like trying to mix oil with water. This milky thing is reversible by the way, just stick it back in 100% ethanol. Also, you can wash out the salycylate by reversing your ethanol steps, and subsequent immunolabelling or intensification steps on re-hydrated ganglia are possible. Keep your eye on your ganglia when you transfer from 100% ethanol to salycylate. If your tissues have some pigment, you may still be able to see them, but if they are like our unpigmented crayfish ganglia, they will become completely invisible. Place them in salycylate on the slides, saves having to feel around for them. Another thing to keep in mind: since this stuff does not harden, you have to be very careful about getting it on objective lenses. It can remove some types of coating. Make sure you wick out as much as possible from under the cover slip. Cover slips can be partially sealed around the edges using Permount or Entellan or any of the other organic based mounting media, but the salycylate will eventually melt through these, so its not permanent. Water bases mounting media do not work, they will be repelled from the salycylate and not seal at all. Store in the freezer if it will not harm your tissues or labels, this stuff will not freeze, but it will eventually evaporate off if kept in the fridge or at room temp. 9) Look under fluorescent microscope with the appropriate filter. Make sure that you don’t overexpose to light to prevent unnecessary bleaching of the dye. Extremely important: keep track of everything you do. Label every dish or tube with your initials and with whatever is distinctive about it (i.e. the primary antibody you are using, etc.). Use the appropriate form, and use a separate form for each preparation. |
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