my cells are human breast cell line GFP/luc but my GFP signal is very low. I cannot find a good anti-GFP to detect it (I also tried TSA amplification, it is not working). Anti-Luciferase antibody from abcam is very unspecific. I tried anti human mitochondrial MAB1273B from millipore and do not get signal. I am thinking to buy the MAB1281 from millipore anti nuclei human, but I read bad comments about this antibody. Any suggestions? Thank you very much in advance.
Hi everybody. I just wanted to comment that I finally get the mitochondrial MAB1273B antibody to work. (in case that someone have the same issue, I like to comment it). HIER with EDTA 1mM - 15mn in pressure cooker, blocking avidin/biotin, and use poly- HRP strep.. it is not perfect but work. I will also do a DNA extraction of the lung met and do a sequencing to confirm human tissu. Thanks in all the case.
Why don't you try over-expression of GFP/Luc followed by injection. I think it would be easier than trying other options that you have explored so far
If your cells are already labelled with Luciferase then why don't you just use the D-luciferin (for firefly luciferase) / coelenterazine (for renilla luciferase) substrate to detect/monitor those cells in vivo by using bioluminescence imaging? What system are you using for the detection of human cells in your xenograft model?
GFP expressions are generally tricky to detect in a mouse model unless your expression is really high. On the contrary, fluorescent proteins towards the red or far red region are comparatively easy to detect since their excitation and emission wavelengths are higher than body/food chow autofluorescence.
Good luck!
Amir
Hi Valentine,
try STEM121 AB (http://)www.stemcellsinc.com/Tools-and-Technologies_SC-Proven-Product-Catalog/Antibody-Reagents/STEM121).
For protocoll:see http://stemcellstm.alphamedpress.org/content/early/2014/12/03/sctm.2014-0078.short?rss=1
In this study we traced human cells in rat tissue, but the protocol also works for mice.
Good luck & kind regards
Darius
Thanks everybody! to reply to murli and amirali, I have overexpressed and sorted my cells for GFP expression before injected into mice. I let the tumor grow for 6months and I was monitored by IVIS, and saw signal. I am now cheking for rare metastase into lung/bone. I saw GFP signal under the scope but I wanted to confirm by IHC the presence of my cell and perhaps some rare dessiminated cells. I will follow the advice of darius, and try to see if anyone have saw expression of this neural markers in my breast cells. Thanks!!!
Hi,
I have had nothing but good experiences in detecting human cells (neurons, fibroblasts) after transplantation in rodents using the human nuclei antibody, MAB1281.
I do not think it labels all human cells but I do not have experience of it not working. I use it 1:200 with amplification.
/Shane
Thanks Shane, I should try this anti-nuclei one also! One question what do you mean by amplification? The TSA kit from perk? Thanks!
Maybe you can try with an other antibody like anti-HLA because you will be specific for human cells.
Since you are using the IVIS system, the best way to monitor cell growth over time would undoubtedly be bioluminescence imaging rather than fluorescence imaging. I would highly recommend you using substrates for the respective luciferace (For firefly luciferace use D-Luciferin and for Renilla luciferase use coelenterazine). The advantage of using bioluminescence over fluorescence would be in terms of better light output, higher sensitivity and no background.
I already monitored my mice for 6month using the Dluciferin and IVIS. I am interested in rare metastases and need to do IHC on the organ of my mice to determine if I have metastases. We usually do GFP staining on lung and get beautiful results. In my experiment i needed to wait more than 6months and I loss some GFP intensity signal and it is why it is tricky. Thanks everybody in all the case!
Intuitively,sSix months seems like a long time for stable transgene expression in the absence of selective pressure on the reporter.
As a first approach, you may consider taking curls or punch biopsies from FFPE tissues and do PCR (target small-amplicons
Hi everybody. I just wanted to comment that I finally get the mitochondrial MAB1273B antibody to work. (in case that someone have the same issue, I like to comment it). HIER with EDTA 1mM - 15mn in pressure cooker, blocking avidin/biotin, and use poly- HRP strep.. it is not perfect but work. I will also do a DNA extraction of the lung met and do a sequencing to confirm human tissu. Thanks in all the case.
Hi Valentine, I tried the MAB1281A4, which is alexa 488 conjugated to detect human cells in mouse brain tissue but it didn't work so far (I tried 1:100 to 1:1000 dilutions). I will try 1:50 before to give up.
Hi Michelangelo,
I never manage to get the anti-nuclei to work, but the anti-human mitochondria does! I recommend you to use the anti human mitochondria MAB1273 (non conjugated like that you can do in green or whatever color...) http://www.emdmillipore.com/US/en/product/Anti-Mitochondria-Antibody%2C-surface-of-intact-mitochondria%2C-clone-113-1,MM_NF-MAB1273
but you need to use HIER with EDTA 15 in pressure cooker to get it work .... if you complain to millipore about your anti-nuclei antibody, you can get the other anti human mitochondria for free.... there are nice with that, we always need to complain :-)
good luck!!!
by the way, just to tell that if your tissue is overfixed, you cannot get it work...
Hi Valentine, what do you mean by overfixed? I'm fighting with MAB1281 at the moment too, but after your comment might give the MAB1273 a go... I just want to make sure first that the tissue is ok for it.
Hi Nora,
when I say overfixed, it is because I have a lung metastasis, that clearly is from my primary site (confirmed by pathologists with H&E stain), but i cannot stained it for my antibodies, I fight as crazy for it, but nothing... some collaborators also tried to do FISH on it and cannot do it... I put my sample in formalin overnight, but it seems that this piece get overfixed..(other sample were fine). but if you already stained with other thing your sample and have signal, your sample should be perfectly fine... I think the MAB1273 is weak so need to have a good sample to begin with.
Good luck!
Val
Hi reader!
a little update concerning this question! In fact the antibody against human HLA (ab70328) work fantastically! it might also work to FACS sort your digested organs in concept... just to let you know and think!
Good luck to all cancer researchers! keep fighting!
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