Cy2, Cy3, and Cy5 Conjugates are brighter in Plastic Mounting Media

Among currently available fluorescent dye conjugates offered by Jackson ImmunoResearch, the cyanine dyes are better able to withstand the harsh dehydration and embedding conditions required for mounting sections in non-polar plastic media, such as DPX and Permount™. Cyanine dyes are brighter in the non-polar environment than in an aqueous medium, resulting in less acquisition time in the confocal microscope than that required for DyLight™ and Alexa Fluor® dyes (Figure 1), even though those dyes are brighter in aqueous mounting media.

The major advantages of plastic over aqueous mounting media are brightness, contrast, and longevity of the fluorescence. Cy2, Cy3, and Cy5 are available conjugated to a selection of secondary antibodies for multiple labeling, streptavidin, and purified IgG controls. For multiple labeling, see the Cy2, Cy3, and Cy5 conjugates in the table below. Figure 2 illustrates the discrete spectra that provide effective separation for multiple labeling. For single labeling protocols that do not require highly cross-adsorbed antibodies, choose Cy3-conjugated antibodies from the lists of whole IgG affinity-purified secondary antibodies. Note that Cy3 conjugates are bright and photostable in the aqueous environment as well as in non-polar media. This is in contrast to Cy2 and Cy5, which are outperformed in aqueous media by Alexa Fluor® conjugates.


 
 
Cy2-goat anti-mouse IgG (H+L), 36.7ms
Alexa Fluor 647-goat anti-rabbit IgG (H+L), 444.8ms

Alexa Fluor 488-goat anti-mouse IgG (H+L), 222.4ms
Dylight 649-goat anti-rabbit IgG (H+L), 444.8ms

Dylight 488-goat anti-mouse IgG (H+L), 222.4ms
Alexa Fluor 647-goat anti-rabbit IgG (H+L), 444.8ms

Figures 1. Sections of gastric mucosa were stained and mounted in DPX . Sections A, B, and C were stained with primary antibodies for abundant antigens (mouse anti-Type IV collagen [green] and rabbit anti-PGP 9.5 [red]). All sections were stained with different fluorophore-conjugated secondary antibodies from Jackson ImmunoResearch. All sections were exposed to light long enough to achieve approximately the same relative brightness. Exposure times are listed in milliseconds (ms). Differences in exposure time for each fluorophore are an indication of its relative degree of brightness. Note that Cy2 conjugates required significantly less exposure time (36.7ms) in DPX than DyLight 488 (222.4ms) and Alexa Fluor® 488 (222.4ms), both with abundant and less abundant (not shown) antigens. Cy2 conjugates also were brighter in DPX than in glycerol mounting media (not shown). Other antigens that are less visible in Cy2-stained sections are not visible with DyLight 488 and Alexa Fluor® 488 (not shown). Similar differences were observed when the other cyanines, Cy3 and Cy5, were compared with corresponding Alexa Fluor® and DyLight dyes. Images and results are courtesy of Dr. Gwen Wendelschafer-Crabb, Kennedy Lab, University of Minnesota. Similar results were reported to us by Dr. Barbara Jones, Department of Neurology and Neurosurgery, McGill University.


Figure 2. Excitation (left) and emission (right) spectra of Cy2 (green), Cy3 (red) and Cy5 (purple). Peak heights were normalized after the spectra were obtained with an M‑series spectrofluorometer system from Photon Technology International, Inc.

Streptavidin and Antibody Description Cyanine
Cy™2
A=492, E=510
Cyanine
Cy™3
A=550, E=57
Cyanine
Cy™5
A=650, E=670

Streptavidin

Streptavidin 016-220-084016-160-084016-170-084

AffiniPure Anti-Chicken

Donkey Anti-Chicken IgY (IgG) (H+L)
(min X Bov, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat, Shp Sr Prot)
ML*703-225-155703-165-155703-175-155

AffiniPure Anti-Goat ***

Donkey Anti-Goat IgG (H+L)
(min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)
ML*705-225-147705-165-147705-175-147

AffiniPure Anti-Guinea Pig

Donkey Anti-Guinea Pig IgG (H+L)
(min X Bov, Ck, Gt, Sy Hms, Hrs, Hu, Ms, Rb, Rat, Shp Sr Prot)
ML*706-225-148706-165-148706-175-148

AffiniPure Anti-Human

Donkey Anti-Human IgG (H+L)
(min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Ms, Rb, Rat, Shp Sr Prot)
ML*709-225-149709-165-149709-175-149

AffiniPure Anti-Mouse

Donkey Anti-Mouse IgG (H+L)
(min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Shp Sr Prot)
ML*715-225-150715-165-150715-175-150
Donkey Anti-Mouse IgG (H+L)
(min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot) **
ML*715-225-151715-165-151715-175-151
Goat Anti-Mouse IgG (H+L)
(min X Hu, Bov, Hrs, Rb, Sw Sr Prot)
ML*115-225-146115-165-146115-175-146
Goat Anti-Mouse IgG (H+L)
(min X Hu, Bov, Hrs, Rb, Rat Sr Prot) **
ML*115-225-166115-165-166115-175-166
Goat Anti-Mouse IgG, Fcγ fragment specific
(min X Hu, Bov, Hrs Sr Prot)
ML*115-225-071115-165-071115-175-071
Goat Anti-Mouse IgG, Fcγ subclass 1 specific
(min X Hu, Bov, Rb Sr Prot)
ML*115-225-205115-165-205115-175-205
Goat Anti-Mouse IgG, Fcγ subclass 2a specific
(min X Hu, Bov, Rb Sr Prot)
ML*115-225-206115-165-206115-175-206
Goat Anti-Mouse IgG, Fcγ subclass 2b specific
(min X Hu, Bov, Rb Sr Prot)
ML*115-225-207115-165-207115-175-207
Goat Anti-Mouse IgG, Fcγ subclass 3 specific
(min X Hu, Bov, Rb Sr Prot)
ML*115-225-209115-165-209115-175-209
Goat Anti-Mouse IgM, µ chain specific
(min X Hu, Bov, Hrs Sr Prot)
ML*115-225-075115-165-075115-175-075

AffiniPure Anti-Rabbit

Donkey Anti-Rabbit IgG (H+L)
(min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot)
ML*711-225-152711-165-152711-175-152
Goat Anti-Rabbit IgG (H+L)
(min X Hu, Ms, Rat Sr Prot)
ML*111-225-144111-165-144111-175-144

AffiniPure Anti-Rat

Donkey Anti-Rat IgG (H+L)
(min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Shp Sr Prot)
ML*712-225-150712-165-150712-175-150
Donkey Anti-Rat IgG (H+L)
(min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Shp Sr Prot) **
ML*712-225-153712-165-153712-175-153
Goat Anti-Rat IgG (H+L)
(min X Hu, Bov, Hrs, Rb Sr Prot)
ML*112-225-143112-165-143112-175-143
Goat Anti-Rat IgG (H+L)
(min X Hu, Bov, Hrs, Ms, Rb Sr Prot) **
ML*112-225-167112-165-167112-175-167
Goat Anti-Rat IgG, Fcγ fragment specific
(min X Hu, Bov, Hrs Sr Prot)
ML*112-225-071112-165-071112-175-071
Goat Anti-Rat IgM, µ chain specific
(min X Hu, Bov, Hrs Sr Prot)
ML*112-225-075112-165-075112-175-075

AffiniPure Anti-Sheep ***

Donkey Anti-Sheep IgG (H+L)
(min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)
ML*713-225-147713-165-147713-175-147

Controls

ChromPure Donkey IgG, whole molecule 017-220-003017-160-003017-170-003
ChromPure Goat IgG, whole molecule 005-220-003005-160-003005-170-003
ChromPure Mouse IgG, whole molecule 
015-160-003

* ML= Multiple Labeling (see Multiple Labeling at www.jireurope.com for an explanation). 
** Caution: See Selection and Location of Affinity-Purified Antibodies before selecting an antibody adsorbed against closelyrelated species.
*** Warning: BSA and dry milk may contain IgG which will react with this antibody. Use of BSA and/or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titer.



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Brands: Jackson ImmunoResearch
Product groups: Antibodies, Chemicals, Proteins / Signaling Molecules