From: "Paul Webster" (Paul_Webster@quickmail.cis.yale.edu)
Subject: Immunogold
If you get gold particles on the formvar film, then there is probably a problem with you gold even before you do experiments. You should not get that sort of background when you do immunolabeling. As for the lack of labeling, if you are repeating an experiment that has worked in the past and everything is the same then it could either be your gold probe or the primary antibody. Primary antibodies and gold probes can deteriorate upon improper storage. The antibodies can aggregate, fall apart or stick to the sides of the tube. With the gold, the most common cause of signal loss is the dissociation of ligand from the gold particles. The free protein (usually protein A of IgG) occupies binding sites but cannot be seen in the microscope (no gold). Protein A-gold is more stable than IgG-gold and is the better all round marker for immunocytochemistry. Another reason for loss of signal is the use of inappropriate bocking agents which either react with the primary atibody or the gold marker (eg. rabbit serum to dilute protein A-gold; fetal calf serum to dilute antibodies to BSA). To give you a better diagnostic we need more details.
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From: Jay Jerome (jjerome@isnet.is.wfu.edu)

In my experience, gold labeling of formvar is usually more indicative of the primary antibody sticking to the formvar. i.e. with the same batches of goat anti-mouse gold we get different levels of formvar background with different primary antibodies. Background is usually highest with antibodies we know to be particularly "sticky". Assuming you are using the gold in an indirect immunostaining protocol, I would suspect that my primary ab had gone bad before blaming the gold. I look forward to hearing other peoples ideas.
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From: Monica Nass (NASSM@QUCDN.QueensU.CA)

I am in complete agreement with Jay Jerome. Optimally, one does not want any g old on the formvar. Its appearance there would indicate high background as a r esult of either a "sticky" primary antibody, as Jay puts it, or of using too mu ch secondary antibody. As long as you've used the same antibody dilutions as b efore, you should get the same results unless some change occurred in either an tibody (or protein A-gold, if you're using it) since the last time you used it.

If you're using protein A-gold conjugate, check the vial to see if the gold has separated from the protein (look for a precipitate at the bottom). Repeated f reezing/thawing will definitely cause this.

If you suspect your primary antibody is at fault, you might try increasing its titre; maybe its activity has not completely disappeared. However I would sugg est obtaining new antibody if possible.
I can't really offer any more since you didn't specifiy what reagents you have used. Hope this has helped.
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FROM: Greg Erdos (gwe@biotech.ufl.edu)

We pay more attention to the background on empty areas of the embedding resin. If those areas are clean we ignore any background on the Formvar. If open areas of the sections show excessive background the above mentioned ideas should be considered.
Also changing blocking protein can help, increasing salt concentration in buffer can help and the addition of Tween 20 can help.

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