Whole IgG antibodies are isolated as intact molecules from antisera by immunoaffinity chromatography. They have an Fc portion and two antigen binding Fab portions joined together by disulfide bonds and therefore they are divalent. The average molecular weight is reported to be about 160 kDa. The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective.
Store freeze-dried powder at 2-8°C. When ready to use, rehydrate with indicated volume of d. water and centrifuge if not clear. Product is stable for about 6 weeks at 2-8°C as an undiluted liquid. Prepare working dilution fresh each day. For extended storage after rehydration, add an equal volume of glycerol (ACS grade or better) for a final concentration of 50%, and store at -20°C as a liquid. Note: after the addition of glycerol, the concentration of protein and buffer salts is one-half of the original. Alternatively, aliquot and freeze the product at -70°C or below in the absence of glycerol. Avoid repeated freezing and thawing.
Expiration date: one year from date of rehydration. However, the expiration date may be extended if the product is stored according to the recommendation and the test results are acceptable for its intended use.
1:100 - 1:400 for most applications
Dilution factors are presented in the form of a range because the optimal dilution is a function of many factors, such as antigen density, permeability, etc. The actual dilution used must be determined empirically.
Cy5 conjugates are excited maximally at 650 nm and fluoresce maximally at 670 nm. They can be excited to about 98% of maximum with a krypton/argon laser (647 nm line) or to about 63% of maximum with a helium/neon laser (633 nm line). Cy5 can be used with a variety of other fluorophores for multiple labeling due to a wide separation of its emission from that of shorter wavelength-emitting fluorophores. However, we recommend Alexa Fluor® 647 as the preferred far-red-fluorescing dye because it is brighter than Cy5 in aqueous mounting media. The reverse is true when mounting in non-polar, plastic mounting media, i.e. Cy5 is brighter than other far-red-fluorescing dyes, including Alexa Fluor® 647 and DyLight 650.
The adult mouse (C57/Bl6) olfactory bulb was stained for astrocytes (blue), microglia (green), and newborn neuroblasts (red). This picture illustrates where the rostral migratory stream, carrying newborn neuroblasts, enters the olfactory bulb (see dense vimentin staining cutting through the center of the picture). Antibodies used: Astrocytes (Vimentin, AB5733 Millipore and
donkey-anti-chicken Cy5, 703-175-155 Jackson ImmunoResearch), Microglia (Iba1, 019-19741 Wako and donkey-anti-rabbit Alexa488, 711-545-152 Jackson ImmunoResearch), and Newborn neuroblasts (marked with BrdU and stained for BrdU, MCA2060T AbD Serotec and donkey-anti-rat Cy3, 712-165-153 Jackson ImmunoResearch).
Carlyn Martina-Mamber, Robin Pronk, Elly M. Hol - Netherlands Institute for Neuroscience
Connexin expression in astrocytes of middle-aged rat retina
Tissue specimen of 9 month old (middle-aged) Wistar rat retina illustrates connexin expression in astrocytes in the mid-peripheral retina. Six-marker immunohistochemistry was executed on retinal wholemount preparations. Connexin 26 (blue, Alexa532-conjugated anti-goat IgG), Connexin 30 (red, Cy™3 AffiniPure Goat Anti-Rabbit IgG (H+L) - Jackson ImmunoResearch 111-165-003), Connexin 43 (green, Alexa 488-conjugated mouse IgG1), Connexin 45 (pale brown, Alexa 594-conjugated anti-sheep IgG), GFAP stained for astrocytes (pale purple, Cy™5 AffiniPure Donkey Anti-Chicken IgY (IgG) (H+L) - Jackson ImmunoResearch 703-175-155), GS Isolectin B4 stained for blood vessels (turquoise, AMCA-conjugated streptavidin - Jackson ImmunoResearch 016-150-084).
Mansour H, McColm JR, Cole L, Weible M II, Korlimbinis A, et al. (2013) Connexin 30 Expression and Frequency of Connexin Heterogeneity in Astrocyte Gap Junction Plaques Increase with Age in the Rat Retina. PLoS ONE 8(3): e57038. doi:10.1371/journal.pone.0057038
This product is for IN VITRO RESEARCH USE ONLY. It is not a medical device and it is not intended for diagnostic or therapeutic purposes.
Cy is a trademark of GE Healthcare Bio-Sciences Ltd. Jackson ImmunoResearch is licensed by GE Healthcare Bio-Sciences Ltd. to manufacture and sell conjugates of Cy2, Cy3, and Cy5 reactive dyes under US Patent Number 5,268,486, and other patents pending.