ibidi Application Guide

Immunofluorescence
AssaysThe Principle of Immunofluorescence
Assays  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 2Immunofluorescence Staining:
A Typical Workflow .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 3Experiment Planning and Sample Preparation  .  .  . 4Sample Fixation .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 4

Immunofluorescence Applied:
Experimental Examples  .  .  .  .  .  .  .  .  .  .  . 13
Rat Hippocampal Neuron and Astrocyte Staining . 14
Visualization of Endothelial Cell Junctions  .  .  .  .  .  . 13
Immunostaining of Rat Dorsal Root Ganglionic
Cells and Schwann Cells .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 13

Cell Permeabilization .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 5

Adherens Junctions and Actin Cytoskeleton of
HUVECs Under Flow .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 14

Blocking .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 5

Mitochondria Staining of MDCK cells .  .  .  .  .  .  .  .  .  . 14

Primary Antibody Incubation .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 5

Focal Adhesions of Differentiated Mouse
Fibroblasts on an Elastic Surface  .  .  .  .  .  .  .  .  .  .  .  .  . 15

Secondary Antibody Incubation  .  .  .  .  .  .  .  .  .  .  .  .  .  . 6Counterstain and Mounting .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 7Microscopy .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 7Troubleshooting .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 8

ImmunofluorescenceWith the ibidi Chambers .  .  .  .  .  .  .  .  .  .  .  . 9Comparison of Immunocytochemistry Protocols .  .  . 10
Chambered Coverslips  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 11
Channel Slides .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 11
Chamber Slides .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 12

Selected Publications
C. Xu, et al. NPTX2 promotes colorectal cancer growth and liver
metastasis by the activation of the canonical Wnt/beta-catenin
pathway via FZD6. Cell Death & Disease, 2019, 10.1038/s41419019-1467-7
read abstract
Kobayashi, T., et al. Principles of early human development andgerm cell program from conserved model systems. Nature,
2017, 10.1038/nature22812
read abstract
H. Tada et al. Porphyromonas gingivalis Gingipain-Dependently
Enhances IL-33 Production in Human Gingival Epithelial Cells.
PloS one, 2016, 10.1371/journal.pone.0152794
read abstract
N. J. Foy, M. Akhrymuk, A. V. Shustov, E. I. Frolova and I.
Frolov. Hypervariable Domain of Nonstructural Protein nsP3 ofVenezuelan Equine Encephalitis Virus Determines Cell-SpecificMode of Virus Replication. Journal of Virology, 2013, 10.1128/
jvi.00720-13
read abstract.com

The Principle of
Immunofluorescence Assays
Immunofluorescence (IF) is a powerful approach forgetting insight into cellular structures and processes
using microscopy. Specific proteins can beassessed for their expression and location, making
immunofluorescence indispensable for scientists tosolve many cell biological questions.
An immunofluorescence experiment is based on thefollowing principal steps:
1.	 Specific antibodies bind to the protein ofinterest.
2. Fluorescent dyes are coupled to these immune
complexes in order to visualize the protein ofinterest using microscopy.
Immunofluorescence staining of the von-Willebrand-Factor(vWF) in endothelial cells (HUVECs). Actin was stained using
phalloidin (green), nuclei are stained with DAPI (blue).

It is distinguished between direct and indirect
immunofluorescence. In direct immunofluorescence,the primary antibody is directly coupled to afluorophore (also called fluorochrome), allowing foreasy handling and quick visualization. In indirect
immunofluorescence, a secondary fluorophorecoupled antibody, which specifically binds to theprimary antibody, is used to visualize the structureof interest.
Although the second approach is more timeconsuming than direct immunofluorescence, it
has several big advantages, such as it is generally
less expensive, because the secondary antibody
can be used for different primary antibodies.
In addition, several proteins can be specificallyvisualized in parallel in one single sample (multicolor
immunofluorescence) by combining multiple primary
antibodies with specific secondary antibodies—
each of them labeled with a different fluorophore.

2

Direct Immunofluorescence

Fluorophore

Indirect Immunofluorescence
Secondaryantibody
Primary
antibody

Antibody
Fluorophore
Antigene

Antigene

The principle of direct immunofluorescence and indirect
immunofluorescence.

Immunofluorescence Staining:
A Typical Workflow
Every immunofluorescence staining protocol consistsof four major steps (cultivation, fixation, staining,
imaging), which can be subdivided as follows:

Experiment Planning
Sample Preparation

Sample Fixation

Cell Permeabilization

Blocking

Primary
Antibody Incubation

Secondary
Antibody Incubation

Counterstain
and Mounting

Microscopy

60 x

Immunofluorescence staining is a very sensitive
method that might require troubleshooting. Slightchanges in the protocol can lead to different resultsthat are no longer comparable. Therefore, it is very
important to precisely maintain the exact same
conditions in your specific protocol (e.g., cell density,
antibody dilution, incubation temperature, andincubation time). The following is an overview of thedifferent steps of an indirect immunofluorescencestaining protocol.

ibidi Solutions
The ibidi μ-Slides and µ-Dishes with a coverslip bottom for inverted microscopy
are available in a variety of geometries that will fit any of your specific needs for
immunocytochemistry. All immunofluorescence staining steps can be performed
directly in the slides or dishes.
The geometry of the ibidi Channel Slides is ideal for the exact exchange of small medium amounts, which isnecessary during immunocytochemistry stainings. In addition, the coverslip bottom of the channel µ-Slides
eliminates the need for additional coverslips.
In the ibidi Chamber Slides, a silicone gasket with separated chambers is mounted on a standard glass
slide. The slides are ideal for the long-term storage of samples that are mounted with a glass coverslip.
Unlike most plastic materials used for cell culture, the ibidi labware is compatible with standard fixation
methods. For a full overview, please check out our chemical compatibility table. All immunofluorescencestaining steps can be performed directly in the slides or dishes.

3

Experiment Planning and Sample Preparation
Targetantigene

Non-target
antigene

Before starting an immunostaining, a literature research should
be done to determine the expression levels and intracellular
localization of the protein of interest in the chosen model system.
Some proteins have a very low expression, either generally, orin certain cell lines. In this case, the expression might have to be
induced by external stimuli or overexpression techniques.
Further, the optimal cell density has to be determined. In
general, a confluence of 70%–80% is recommended for IF.
In addition, the ideal cell culture vessel geometry and substrate/coating must be determined, and needs to be compatible withthe chosen microscopy method. During the whole experiment, itis crucial that the cells never dry out, which should be consideredwhen choosing the appropriate cell culture vessel geometry.
Finally, the number of samples to be stained and analyzed forstatistical significance, including the appropriate controls, shouldbe planned in advance.

Sample Fixation
The first step of an immunofluorescence staining protocol is to
fixate the sample. This is usually done by incubating the sample
for 10 minutes at room temperature in a 4% formalin solution(in PBS, pH 7.4), which crosslinks the proteins. The sample canalso be fixated in 100% chilled methanol or acetone.
Optimal fixation conditions should be determined individuallyfor each experiment. Some proteins are destroyed by methanol
fixation, and some antibodies do not detect proteins in formalinfixated samples. After fixation, it is very important to wash thesample 3x for 5 minutes in a washing solution, (e.g., PBS) to
remove the fixation solution completely.

Cell Permeabilization
In order to stain intracellular proteins, the cell needs to bepermeabilized. Without this step, it is not possible for the antibodies
to enter the cell through the lipid membrane. The permeabilization
requires incubation in a detergent, for example Triton X-100 or
Tween-20 (for a less harsh permeabilization) in a PBS solution.
This step must be optimized depending on the protein of interest,
the used antibody, and the experimental conditions. Especiallywhen staining membranous proteins, the permeabilization step
has to be done with caution, because Triton X-100 can destroy
the cell membrane. In this case, using saponin might be analternative to classic detergents. Don’t forget that methanolfixed samples are already permeabilized, since alcohols easily
wash out the lipids of the cell membrane.
After this step, the sample has to be washed 3x for 5 minutes ina washing solution.

4

BlockingTargetantigene

Blockednon-target
antigene

In order to minimize intra- or extracellular background signals,
non-specific antigens should be blocked by incubating thesample in (1) the serum of the host, in which the secondary
antibody was made, (2) bovine serum albumin (BSA), or (3)milk. The first option is the most recommended because of its
highest specificity.
Typical blocking times are from 30 minutes up to one hour.
Excessively long blocking steps should be avoided, as thiscan reduce the specific binding of the primary antibody, andtherefore reduce the signal.

Primary Antibody Incubation
The selection of the primary antibody and its incubation
conditions is the most critical step of an immunofluorescencestaining protocol. Especially, if the protocol has not yet been
established in the lab, a literature research is crucial. A suitableprimary antibody must have a high specificity for the antigen
of interest. Furthermore, whether the antibody is mono- or
polyclonal influences its specificity. Many antibody manufacturers
list references on their product pages, in which the antibody was
successfully used in an immunofluorescence staining.
Another highly relevant property of the primary antibody is its
originating host, as it determines the secondary antibody. This isespecially important when doing multicolor stainings, because
different specific primary antibodies for the parallel detectionof several antigens in the same sample must be produced indifferent hosts, in order to avoid cross-reactivity.
The optimal incubation conditions must be determined carefullyfor each experiment. Antibody concentrations that are too highand incubation times that are too long can result in an unspecificbackground signal. Conversely, concentrations that are too low
and incubation times that are too short can lead to a very weakor missing signal.
After incubation with the primary antibody, the samples should
be washed 3x for 5 minutes in a washing solution in order toavoid background fluorescence.

5Secondary Antibody Incubation
In standard immunofluorescence assays, the secondary
antibody is conjugated to a fluorophore, which emits lightwhen excited at a defined wavelength. The secondary antibody
specifically binds to the first antibody. Therefore, it is essentialthat the secondary antibody is specific to the host in which theprimary antibody was produced. For a successful experiment,
the chosen microscopy technique and the equipment (filters,
lasers, cameras, and detectors) also need to be compatible withthe chosen fluorescence dye.
To be suitable for fluorescence microscopy, stable fluorophores
must be used, because the sample is generally exposed to ahigh number of photons. Also, fluorophore brightness should
be taken into account, because the antigen with the highest
expression level should be detected with the fluorophorewith the lowest brightness. When doing a multicolor staining,
the spectral overlap of the conjugated fluorophores should be
carefully considered as well. Respective tables can be found on
the manufacturers’ websites.
The incubation in the secondary antibody solution should
be carried out according to manufacturer’s protocol. Since
fluorophores are sensitive to light, from now on all steps of the
protocol need to be carried out in the dark.
In order to avoid background fluorescence, the samples
should be washed 3x for 5 minutes in a washing solution after
incubation with the secondary antibody.

Excurse: What is a fluorophore?
Energy levels

S2

Radiation-freeenergy transfer

Excitation

Emission

S0

Ground state

Intensity

Stokes
shift

S0S2

S1S0

Wavelength [nm]6

Excited states
S1

A fluorophore (also called fluorochrome) is a chemical molecule,which is used to label specific structures for microscopic
analysis. For example, the green fluorescent protein (GFP), redfluorescent protein (RFP), and yellow fluorescent protein (YFP)
are widely-used fluorophores in research.
Fluorophores are excited when they absorb light of a certain
wavelength, leading to re-emission of light at a defined higher
wavelength. A successful excitation by light transfers thefluorophore from the ground state (S0) to an excited state (S2).
This high-energy state is not stable and quickly undergoeschanges. The fluorophore vibrates, leading to light releaseand a return to the ground state after a few picoseconds.
The vibrations in the exited states cause the so-called Stokes
shift, which means that the wavelength of the emitted light isalways higher than that of the excitation light.

Counterstain and Mounting
The final step before microscopy is the counterstaining of
the nuclei and the mounting. In order to avoid drying out of
the samples, and to guarantee a stable refractive index of thecellular environment (a prerequisite for successful microscopy),
the sample needs to be mounted.
For this, the sample should be covered with a mounting mediumthat has a low autofluorescence. DAPI, the standard for nuclear
counterstaining, is either included in the mounting medium, or
can be added separately.

Microscopy
To get optimal results, the microscopic analysis of the immunofluorescence staining should be done directly after the mounting.
Many microscopy techniques exist, each optimized for different
experimental approaches. For standard immunofluorescencestainings, epifluorescence and confocal microscopy are widelyused methods. Of course, all parameters such as magnificationand exposure should be carefully and individually determined.

60 x

Please find a detailed overview of the different microscopy
techniques and their applications on our website, or download
the “Microscopy With the ibidi Chambers” Application
Guide as a PDF.

ibidi Solutions
The ibidi Mounting Medium and the ibidi Mounting Medium With DAPI have avery low autofluorescence, prevent photobleaching and allow the sample to be stored
for several weeks on the µ-Slide without the need for additional coverslips.
If your experiment requires long-term storage of immunostained samples, we recommend
using the ibidi Chamber Slides, removable. Since signals could fade if fluorophores
are exposed to light for a longer period, the samples always must be stored in the dark.

7Troubleshooting
Immunocytochemistry is a very sensitive method that requires a lot of experience and optimization. Slightchanges in the protocol can markedly alter the results. If you get a low signal, no signal, or a high background,please consult the following troubleshooting guide.

High Background
ReasonSolution

Inappropriate or too long fixation, leading to artefacts Reduce fixation time or change the fixative
Insufficient blocking

Prolong the incubation time, consider using a different
blocking solution (we recommend using serum from thesecondary antibody host)No specificity of the primary antibody

Use a primary antibody that is proven to work for immunofluorescence in the chosen model system; if available, use aknockdown/knockout sample as a negative control

Too high primary/secondary antibody concentration, Optimize the antibody concentration and incubation time/
too long incubation time, too high incubation temperature temperature, consult manufacturer’s protocol
Cross-reactivity of the secondary antibody

Use isotype controls for the secondary antibody to checkfor cross-reactivity

Not enough washing

Verify that all washing steps are carried out properly; ifnecessary, prolong the washing steps

Bleaching during imaging

Use a secondary antibody conjugated to a fluorophore
suitable for your chosen microscopy technique

Low signal intensity, resulting in noise

Optimize the signal-to-noise ratio, e.g., by using abrighter fluorophore for detection; if applicable, increase
the expression of the antigen of interest (e.g., byoverexpression or by addition of inducing agents)

High autofluorescence

Check the sample autofluorescence by using unstained
controls; use fresh fixation solutions (expired formalin solutions
might have a high autofluorescence); use materials with low
autofluorescence (e.g., ibidi µ-Slides or Chamber Slides);use mounting medium with low autofluorescence (e.g., ibidi
Mounting Medium / ibidi Mounting Medium With DAPI)Low Signal or Lack of Signal

8

ReasonSolution

Drying out of the sample

Always keep the sample moist

Overfixation of the sample, leading to epitope
damageReduce fixation time or change the fixative

Inadequate permeabilization method

Optimize or skip the permeabilization step

No binding of primary antibody to the antigen ofinterest

Use a primary antibody that is proven to work for
immunofluorescence in the chosen model system; checkthe antibody functionality by using a positive control (e.g.,by overexpression or by addition of inducing agents)

Too high primary/secondary antibody dilution,
too short incubation time, too low incubation
temperature

Increase the antibody concentration or the incubation time/
temperature; consult manufacturer’s protocol

Very low or no antigen expression

Use a positive control (e.g., an overexpression model);reconsider your experimental system

Inappropriate microscopy detection method

Use a more sensitive method for image acquisition; checkyour used filter/laser setup; use a brighter fluorophore fordetection

ImmunofluorescenceWith the ibidi Chambers
ibidi provides several solutions that fit your needs
for immunofluorescence assays:

Bottom material

Chambered Coverslips

Channel Slides

Chamber Slides, removable

Glass Coverslip
or Polymer Coverslip

Glass Coverslip
or Polymer Coverslip

Standard glass slideNo

Yes (for mounting)

Additional coverslips No
required?
Microscope type

Inverted

Inverted

Inverted & upright

Mounting medium

Non-hardening

Non-hardening

Hardening

Sample storage

Short-term

Short-termLong-term

Benefits of ibidi µ-Chambers
1. High-resolution imaging
The ibidi slides are ideal for widefield fluorescence, confocal imaging, FRAP, FRET, FLIM,and undisturbed phase contrast imaging.
2. Fast and simple handling
The all-in-one chambers simplify yourimmunofluorescence protocol.

With their thin coverslip
botton, the ibidi µ-Dishes
and µ-Plates are also
ideally suitable for immunofluorescence stainings andhigh-resolution microscopy.

3. Cost-effective experiments
Only a small number of cells and low reagent
volumes are needed.

Find detailed immunofluorescence staining protocols in the ibidi Application Notes:• AN 02: Fluorescence Staining using a µ-Slide I (PDF)• AN 09: Fluorescence Staining using a µ-Slide VI 0.4 (PDF)• AN 15: Fluorescence Staining using a µ-Slide y-shaped (PDF)• AN 16: Fluorescence Staining using a µ-Slide 8 Well (PDF)• AN 49: Fluorescence Staining using a 12 Well Chamber, removable (PDF)• AN 50: Fluorescence Staining using a 3 Well Chamber, removable (PDF)• AN 45: Mounting Medium Types (PDF)

9Comparison of Immunocytochemistry Protocols:
Traditional Staining vs. Staining With ibidi Solutions
When using any of the ibidi solutions, the immunofluorescence staining protocol is much shorter than the
traditional protocol. There is no need to grow the cells on loose coverslips—the cells can be grown and stained
directly in the ibidi slides.

Protocol With Cells on Coverslips
Traditional method with nail polish mounting

Protocol With ibidi µ-Slides
Time-saving method using all-in-one chambers• Sterilize coverslips and slides• Sterilize coverslips and slides• Coat coverslips• Coat coverslips• Place sterile coverslips into 6-well plate• Place sterile coverslips into 6-well plate• Seed cells in large volume• Seed cells in large volume• Peel off the coverslip• Peel off the coverslip• Wash• Wash• Fix – wash – permeabilize – wash – block• Fix – wash – permeabilize – wash – block• Incubate in primary antibody – wash – incubate
in secondary antibody• Incubate in primary antibody – wash – incubate
in secondary antibody• Wash• Wash• Mount cells with mounting medium• Mount cells with mounting medium• Mount coverslip with nail polish• Mount coverslip with nail polishibidi Solutions
The ibidi Mounting Medium and the ibidi Mounting Medium With DAPI have avery low autofluorescence, prevent photobleaching and allow the sample to be stored
for several weeks on the µ-Slide without the need for additional coverslips.
If your experiment requires the long-term storage of immunostained samples, werecommend using the ibidi Chamber Slides, removable.

10Chambered Coverslips

Cell seeding

Cultivation

Fixation

Staining

Imaging

60 x

When using chambered coverslips, such as the ibidi µ-Slides, the cell culture and the whole immunofluorescencestaining protocol can be performed in the same well, without the necessity of any additional coverslips or tweezers.
After mounting, the results can be observed through the coverslip bottom using high-resolution microscopy.

Advantages

Limitations• No coverslip handling• Storage time is restricted to several weeks,
because of the gas exchange through the plastic
material from which the slides are made• Parallel assays without cross-contamination

Find more information and technical details about
the coverslip bottom of the ibidi chambers on
our website.

The ibidi µ-Slides areavailable in a variety of wellnumbers, volumes, andgeometries that fit yourspecific experiment.

Channel Slides

Cell seeding

Cultivation

Fixation

Staining

Imaging

60 x

The ibidi channel slides are particularly suitable for immunofluorescence stainings: The geometry of the ibidi Channel
Slides is ideal for the exact exchange of small medium amounts, which is necessary during immunocytochemistry
stainings. In addition, the coverslip bottom of the channel µ-Slides eliminates the need for additional coverslips.

Advantages

Limitations• Meniscus-free phase contrast microscopy• Storage time is restricted to several weeks,
because of the gas exchange through the plastic
material from which the slides are made• Low volumes of reagents needed• Homogeneous cell and antibody distribution

Find more information and technical details about
the coverslip bottom of the ibidi chambers on
our website.

The µ-Slide VI 0.4 is ideal for
general low-volume immunofluorescence assays.
The µ-Slide I Luer enables
immunofluorescence staining
in low-density cultures in thecontext of flow experiments.

11

Chamber Slides
60 x

Seeding

Cultivation

Chamber removal

In the ibidi Chamber Slides, a silicone gasket withseparated chambers is mounted on a standard
glass slide. The ibidi Chamber Slides for immunofluorescence are ideal for the long-term storage ofsamples that are mounted with a glass coverslip.

Fixation & Staining

Mounting & Imaging

Storage

Cultivation
Lid
Removable silicone
gasket (wells)
Standard glass slide

For high-throughput approaches, the silicone gasket
can be removed before starting the staining. This allows
100 mlfor the processing of several
slides at once in a staining
jar. As an alternative to lowthroughput experiments, thesilicone gasket can be left inposition, allowing for separate
staining in each well.

60 x
Glass coverslip
Mounting medium
Standard glass slide

Advantages

Limitations• Ideal for long-term storage• No high-resolution microscopy during cell
cultivation• High-throughput screening possible

The ibidi Chamber Slides,
removable are available
with 3 wells, 8 wells, and12 wells.

12

MicroscopyImmunofluorescence Applied:
Experimental Examples• Visualization of Endothelial Cell Junctions• Immunostaining of Rat Dorsal Root Ganglionic Cells and Schwann Cells• Rat Hippocampal Neuron and Astrocyte Staining• Adherens Junctions and Actin Cytoskeleton of HUVECs Under Flow
• Mitochondria Staining of MDCK cells• Focal Adhesions of Differentiated Mouse Fibroblasts on an Elastic Surface

Visualization of Endothelial Cell Junctions
Immunofluorescence staining of primary mousebrain microvascular endothelial cells (pMBMECs),
cultured in the ibidi 12 Well Chamber, removable.
Red color delineates the endothelial cell junctions
labeled for zonula occludens (ZO)-1, highlighting
their matured state. Blue color shows the nuclei
stained with DAPI. The image was acquired with a
20x objective on a Nikon Eclipse microscope.
Data by Sidar Aydin, Britta Engelhardt, Ruth Lyck, Theodor
Kocher Institute, Bern, Switzerland.

ibidi product used:

12 Well Chamber, removable

Immunostaining of Rat Dorsal Root Ganglionic Cells and Schwann Cells
Rat dorsal root ganglionic cells and Schwann cells
cultured in an ibidi μ-Slide 8 Well and stained forneurofilament (green), NGFR (magenta), and DAPI
(white). The image was obtained with a LEICA SP8X
laser scanning microscope.
Data by Tamara Weiss, Division of Plastic and Reconstructive
Surgery, Medical University of Vienna, Austria.

ibidi product used:μ-Slide 8 Well

13

Rat Hippocampal Neuron and Astrocyte Staining
Confocal microscopy of rat hippocampal neurons
plated over astrocytes, cultured on ibidi µ-Plate
24 Well. Red is Synapsin 1, a pre-synaptic marker,
white is neuron-specific Tubulin Beta-III, and blue is
DAPI, a nuclear counterstain.
Data by Daniel Hoeppner, Lieber Institute for Brain
Development. Baltimore, MD, USA.

ibidi product used:μ-Plate 24 Well

Adherens Junctions and Actin Cytoskeleton of HUVECs Under Flow
Human umbilical vein endothelial cells (HUVECs)
were cultured under flow conditions in the ibidi
μ-Slide I 0.4 Luer.
Red: actin cytoskeleton (Cy5-conjugated antibody).
Green: adherens junctions, marked by VE-cadherin(Alexa 488-conjugated antibody).
Blue: nuclear counterstaining (DAPI).
Data by S. Zahler, Munich, Germany.

ibidi product used:μ-Slide I 0.4 Luer

Mitochondria Staining of MDCK cells
Madin-Darby canine kidney (MDCK) cells were
cultured in the ibidi μ-Slide VI 0.4 .
Green: F-actin cytoskeleton (Alexa488-Phalloidin).
Red: mitochondria (MitoTracker).
Blue: nuclear counterstaining (DAPI). Widefield fluorescence using 100x objective lens with oil immersion.
ibidi product used:μ-Slide VI 0.4

14Focal Adhesions of Differentiated Mouse Fibroblasts on an Elastic Surface
Differentiated mouse fibroblasts were cultured onan elastic surface using the µ-Dish 35 mm, high ESS(28 kPa). Green: focal adhesions, marked by zyxin(Alexa 488-conjugated antibody). Red: alpha-smoothmuscle actin (Cy5-conjugated antibody).
Data by R. Merkel, Jülich, Germany.

ibidi product used:µ-Dish 35 mm, high ESS

15

16

Manufacturer

North American Headquarters

ibidi GmbH
Lochhamer Schlag 1182166 Gräfelfing
Germany

ibidi USA, Inc.
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Fitchburg, WI 53719
USA

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Fax:
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only! Errors and omissions excepted.

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