A Troubleshooting Tale

This post is for Physioprof, because he loves this shit. The rest of you will most likely find it to be terminally boring. Fair warning.The problem:BullshitExperiment requires final validation using FavoriteAntibody. Recent attempts to use FavoriteAntibody on tissue prepared in such manner as for BullshitExperiment have resulted in no signal from FavoriteAntibody either on experimental tissue or positive controls. Oh crap.The hypotheses:FavoriteAntibody is failing because it's old. The problem is not with FavoriteAntibody but with secondaries.There is a fatal flaw in BullshitExperiment which causes cells not to express FavoriteAntigen.Strategy #1:To address hypothesis #1, I used the same protocol as I do for FavoriteAntibody, but used LabCommunalAntibody as the primary instead. LabCommunalAntibody is also usually very good, is used with consistent success by several other lab members, is raised in the same host species as FavoriteAntibody, and detects cells in my tissue of interest (though different cells than those which label with FavoriteAntibody).Result #1:No signal with LabCommunalAntibody (Uh-oh).Conclusion #1:While this does not tell me for sure that FavoriteAntibody is old and no longer useful, it is unlikely that both FavoriteAntibody and LabCommunalAntibody are both facing the same fate. This result suggests that the problem is more likely to be with the secondaries or a fatal flaw in the experiment.Strategy #2:Apply FavoriteAntibody to positive control tissue in 3 groups. Each group will contain 4 slides (3 with primary antibody incubation and 1 without). Each group will be incubated in a different secondary: goat anti-rabbit 488, goat anti-rabbit 546, donkey anti-rabbit 546. FavoriteAntibody is raised in rabbit and I frequently use it for double-labeling, so I have had success with all of these secondary antibodies in the past.Result #2:goat anti-rabbit 488 - strong signal, no background, no signal without primarygoat anti-rabbit 546 - strong signal, no background, no signal without primarydonkey anti-rabbit 546 - inconsistent signal (some slides show some cells with comparable signals to other secondaries but other cells with weak signal, some slides show uniformly weak signal), no background, no signal without primaryConclusion #2: Donkey anti-rabbit 546 gives inconsistent label on my tissue of interest when used with FavoriteAntibody. Goat is where it's at, yo! This indicates that my previous problems may have been attributable to use of the donkey secondary but does not yet eliminate the possibility that there is a fatal flaw in BullshitExperiment which causes cells not to express FavoriteAntigen.Strategy #3:Apply FavoriteAntibody (with goat secondary!!) to experimental tissue of interest, as well as to age-matched controls (sham manipulation), and to positive control tissue that has given me good signal in Strategy #2.Results/Conclusions #3:I'll let you know.Even if I get the label to work in BullshitExperiment, this will not alleviate the general bullshit issues with this experiment, like the fact that negative results will be inconclusive. But if I get all this done before meeting with GrAdvisor I have a much stronger case for dropping BullshitExperiment from the ManuBeast, and the added bonus of not having lost faith in my FavoriteAntibody, which will be used for many upcoming experiments. Baby steps.

Labels: Troubleshooting