I also followed the storage instructions for the secondary antibody (ab60314) shown in both the datasheet that the antibody came with and the Abcam website for this particular antibody so I've kept it at 4C. When I first got my antibodies, I followed the storage instructions for all of them therefore I aliquoted both: the primary antibody (ab83174) and the Rabbit Isotype Ctrl (ab37406) and then I stored them at -20 C. As I'm not an expert on the art of performing ICC nor in biology, I've been reading about those things that might potentialy affect the staining and I've noticed that the majority of the antibodies are aliquoted and stored either at -20 or -80 C. The samples are going to be incubated with the primary antibody overnight at 4C and hopefully the specific signal from the positive control will be better. At the moment I'm staining my last batch of samples (fixed with 50% methanol and 50% acetone). And I'll also grow the cell line of interest (Caki-2) and perform the staining on the glass they've growth on so we can conclusively know whether the trypsin is having some detrimental effect on the staining. I'll run the experiment you suggested next week (as soon as I get some HeLa cells). Unfortunately the secondary antibody hasn't been used with another primary antibody and the primary antibody hasn't been detected by another secondary antibody -this is the only system I currently have, but I do see the point of checking the actual effectiveness of both so I'll try to get some aliquotes from my collegues and see what sort of staining profile I get. The ration cells per area is: 12,500 cells/cm2 (which has demonstrated by previous studies that enable the log growth of the cells). Also, the cells are seeded at a concentration of 40,000 cells/ml (concentration that has shown to give place to a log growth).

The cell viability is checked by means of the trypan blue exclusion method, about 1-2% of cells are dead as they don't efflux effectively the dye, therefore I've confirmed in every sample prep that the cells are viable. Unfortunately I have no data supporting the expression levels of the CDT1 in this particular cell line so it's definitely worthwhile to stain HeLa cells and see whether the weak fluorescent signal arises due to the low concentration of CDT1 within my cells. It is highly expressed in lung, colon, placenta, testis, pituitary gland and cerebellum, MDA-MB435 cell line If you need any further assistance in the future, please do not hesitate to contact me. Isotype controls: can be searched at this site: ab36204, ab48614, ab66219, ab102998 -> Rabbit IgG - ab37406, ab125938, ab128142 ab83508 -> Mouse IgG1 - ab91353, ab27479, ab81032 Positive control: ab36204 - CRMP4 - It is mainly expressed in heart and skeletal muscle and also strongly expressed in fetal brain and spinal cord (human, mouse or rat), ab48614 -Ceruloplasmin - It is expressed by the liver and secreted in plasma, ab66219 - Oprl1 - Brain (cortex), ab83508 - NG2 - It is detected only in malignant melanoma cells, SK-MEL-28 cells, ab102998 -B3GNT5- only tested in WB - Widely expressed. However, I can provide a range of the IgG: 10-15 mg/ml. The other antibody ab36204 (Lot: GR42382) is not affinity purified but whole antiserum so the IgG is not quantified. I can confirm the concentration of ab66219 (Batch: GR9812) of this ab is 1 mg/ml. Int J Physiol Pathophysiol Pharmacol 3:97-106 (2011). Exercise mitigates homocysteine - ß2-adrenergic receptor interactions to ameliorate contractile dysfunction in diabetes. Perivascular Mesenchymal Progenitors in Human Fetal and Adult Liver. Modular extracellular sensor architecture for engineering mammalian cell-based devices. Behavior of multipotent stem cells isolated in mobilized peripheral blood from sheep after culture with human chondrogenic medium. Band 3 ectopic expression in colorectal cancer induces an increase in erythrocyte membrane-bound IgG and may cause immune-related anemia. Publishing research using ab37406? Please let us know so that we can cite the reference in this datasheet.Īb37406 has been referenced in 5 publications.

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