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STED Microscopy Sample Preparation

Chromeo™ STED Immunofluorescence Systemproperly prepared samples ensure your STED microscopy yields clear, conclusive high-resolution images

Fluorescent Microscopy with fluorescently labeled antibodies is widely used to determine the sub-cellular
localization of specific proteins and to answer questions with regards to protein modification, interactions
and life cycle. However, the resolution attainable in immunofluorescence experiments has until recently been
limited by a very specific physical property known as the Abbe Law of Diffraction Limiting Resolution. The Abbelimit restricts the ability of the observer to visually resolve objects separated by less than ~200 nm. However, recent
advances in super-resolution techniques such as STED (STimulated Emission Depletion) in combination with confocal
scanning enable the observer to exceed the Abbe limit and resolve details as small as 20 nanometers. This facilitates
the imaging of sub-cellular structures that previously could not be visualized.

Figure 1: STED microscopy overcomes the Abbe limit and enables a much higher level of resolution than confocal microscopy.
HeLa cells were stained with alpha Tubulin mouse monoclonal antibody (Clone 5-B-1-2) and Chromeo 494 Goat anti-mouse IgG (Catalog No. 15032). Histone H3 was stainedwith Histone H3 K4me3 rabbit polyclonal antibody (Catalog No. 39159) and the ATTO 647N STED Goat anti-rabbit IgG (Catalog No. 15048) secondary antibody. The left image
was prepared using a confocal microscope, while that on the right was produced using a STED microscope. Images courtesy of Leica Microsystems, Germany.

Proper sample preparation ensures high-quality images
For super-resolution microscopy to yield
clear, conclusive high-resolution images, it
is extremely important to optimize thetechniques and reagents used for sample
preparation. Proper sample preparationis among the most significant factors for
obtaining high-quality images. To help ensurethat you consistently achieve the best results
possible, Active Motif collaborated with
Leica Microsystems to develop the Chromeo™
STED Immunofluorescence System. This kit

helps take the guesswork and challenge outof preparing samples for STED microscopy
by providing a complete set of proven,
QC-tested reagents and an optimized protocol. In addition to certifying this kit for use
with its STED microscopes, Leica has tested
and recommends many of Active Motif’s
fluorescent dyes and fluorescent secondary
antibody conjugates for use with its instruments because they meet the specifications
required for STED microscopy (see page 2).

Advantages of using theChromeo™ STED System• Optimized reagents and protocol help
ensure proper sample preparation,
resulting in the highest quality images• Certified and recommended byLeica for use in STED microscopy• Includes optically matched coverslipsfor maximum resolution
For more complete details, please give us acall or visit us at www.activemotif.com/sted.

North America 877 222 9543 Europe +32 (0)2 653 0001 Japan +81 (0)3 5225 3638

STED Microscopy Sample Preparationwww.activemotif.com

Fluorescent Secondary Antibodies Validated for STED Microscopy
Chromeo™ 488 and Chromeo™ 505 for CW STED microscopy
Leica Microsystems has certified Active
Motif’s Chromeo™ 488 and Chromeo™ 505 fluorescent dyes and secondary antibody conjugates for use with its TCS STED CW microscope, which uses a continuous argon gaslaser (488 nm and 515 nm) for excitation anda continuous 592 nm fiber laser for depletion. The fluorescent properties of Chromeo
488 and Chromeo 505 meet the specifications required to perform STED microscopy

with the continuous lasers, and enable live
cell imaging below 80 nm. In addition to thevalidated, high-quality secondary antibody
conjugates, both dyes are available as reactive NHS-Esters and as Carboxylic Acids.

ATTO (STED) & Chromeo™ 494
for TCS STED microscopy
The Leica TCS STED system utilizes a pulsed
640 nm excitation laser combined with a750 nm depletion laser, enabling it to reach aspatial resolution of 50-70 nm. For use in thiswavelength range, Leica Microsystems recommends Active Motif’s fluorescent ATTO 647N
(STED) and ATTO 655 (STED) secondary antibody conjugates. These ATTO dye conjugateshave been maximally cross-adsorbed against
IgG’s of a variety of species to eliminate
background caused by non-specific binding.
The TCS STED microscope can also beupgraded for dual color STED simply byintegrating a second, 531 nm excitation laser.
This enables use of a second dye in highresolution STED microscopy. With dual color
STED microscopy, co-localization of proteinscan be studied in a novel and reliable way.
Chromeo™ 494 fluorescent dye and secondary antibody conjugates have been certified
by Leica Microsystems for use with either
ATTO conjugate in dual color TCS STED.
Because Active Motif’s Fluorescent Antibody
Conjugates have been prepared using an
optimized protocol that ensures the highest
fluorescence intensity and stability, they canbe used in such demanding applications.
CONTENTS & STORAGE

The Chromeo™ STED Immunofluorescence System
contains sufficient reagents to for the preparation of
24 immunofluorescence slides and includes MAXblock™
Blocking Medium, MAXbind™ Staining Medium,MAXwash™ Washing Medium, MAXfluor™ Mounting
Medium S, 24 MAX Stain™ Slides and 50 MAX Stain™
Coverslips. Reagent storage conditions vary from room
temperature to 4°C. All reagents are guaranteed stable for6 months from date of receipt when stored properly.

Figure 2: Chromeo 488 antibody conjugates in CW STED microscopy.
Nuclear pore protein-1 (NUP-1) was stained with a primary monoclonal mouse antibody and with Chromeo 488Goat anti-mouse IgG (Catalog No. 15031) secondary antibody (left). Vimentin was stained with a primary polyclonal
rabbit antibody and with Chromeo 488 Goat anti-rabbit IgG (Catalog No. 15041) secondary antibody (right). These
STED images are courtesy of Leica Microsystems, Germany.

Dye

Absorption

Emission

STED System

Chromeo™ 488 IgG

498 nm524 nmTCS STED CW

Chromeo™ 505 IgG

514 nm

530 nmTCS STED CW

Chromeo™ 494 IgG

489 nm624 nmTCS STED (dual color)

ATTO 647N IgG

644 nm

669 nmTCS STEDATTO 655 IgG

663 nm

684 nmTCS STED

Table 1: Properties of Active Motif fluorescent secondaries certified for use in STED by Leica Microsystems.

ProductFormat

Catalog No.

Chromeo™ STED Immunofluorescence System

1 kit

15260

Chromeo™ 488 Goat anti-Mouse IgG
Chromeo™ 488 Goat anti-Rabbit IgG

1 mg
1 mg

15031
15041

Chromeo™ 505 Goat anti-Mouse IgG
Chromeo™ 505 Goat anti-Rabbit IgG

1 mg
1 mg

15030
15040

Chromeo™ 494 Goat anti-Mouse IgG
Chromeo™ 494 Goat anti-Rabbit IgG

1 mg
1 mg

15032
15042

ATTO 647N (STED) Goat anti-Mouse IgG
ATTO 647N (STED) Goat anti-Rabbit IgG

250 µl250 µl

15038
15048

ATTO 655 (STED) Goat anti-Mouse IgG
ATTO 655 (STED) Goat anti-Rabbit IgG

250 µl250 µl

15039
15049

North America 877 222 9543 Europe +32 (0)2 653 0001 Japan +81 (0)3 5225 3638