jaimev

So our lab suddenly has been having issues with IHC. About a month ago, everything was working fine, but over the last several weeks; we began to notice bright flecks in our fluorescent reactions. Sometimes the fluorescence appears to clump in the tissue and other times it looks very speckled. Along with this, we see poor labeling with antibodies that have been shown in the past to work just fine. I began checking our reagents and found bacterial contamination of our PBS. Unfortunately, someone in our lab contaminated the stock solution and I hadn't noticed. However, we have tried making new PBS, using brand new plates, autoclaving glass vials used for incubating antibodies overnight, etc. But we still can't seem to get our IHC back to normal. I figured the antibodies maybe had contamination so I tried another lab's secondary antibody (thinking our glycerol was contaminated too), but it looked all weird too! Our lab is low on funds so my PI doesn't want to just buy all new antibodies without knowing if they are the problem. Has anyone had something like this happen to their IHC and is the flecked/clumpy secondary a contamination issue or is this something else I am not even thinking about? Any help would be appreciated!

Thanks!

So I have been trying to optimize PCR conditions for the last few months and have minimal experience with PCR. Additionally, we don't have anyone in lab with experience so I apologize if this is a basic question. I recently discovered a problem after having several successful PCR optimization results. I use PCR to look at 5-HT1A receptor levels in rat brains, but wanted to start off showing knockdown in cells (this is just regular RT-PCR not qRT-PCR). Prior to recently, all of my controls have been working just fine. I then suddenly started getting bands in my negative control RIGHT at the level of my band of interest (500bp so probably not primer dimers). So I used fresh aliquots of everything and still a faint band. I checked my loading of the gel by placing the negative control by itself, but still a faint band. Then I tried using new water from another lab with no luck. Finally, I then tried running two negative controls; one in which there was water and primers with mastermix and also one that had only water and mastermix but no primers. The sample without primers had no band while the primer negative control did! Interestingly enough, my untransfected cells (which had primers) also don't have this band. So if it was linked to my primers, wouldn't I see it in my untransfected cells? I have a picture I can also try uploading later, but basically I was wondering if anyone knows what I would get amplification in my negative control with primers, but not with untransfected cells (which had primers) and a negative control without primers!? Any information would be greatly appreciated. Trying to illustrate knockdown of this receptor will be the death of me!


Ok. So I loaded the picture and I know that I have some non-specific amplification higher up (which is also new), but I don't want to get into that at the moment. The last band in the ladder is 500bps which is where my band of interest is located. The order of my samples is as follows:

1) ladder
2) untransfected cells
3-7) transfected cells with my receptor
8) negative control with primers
9) negative control without primers
10) positive control with plasmid DNA

 Negative control woes.jpg   29.83K   191 downloads