Sample preparation for light
microscopy
cellim@ceitec.muni.cz
https://cellim.ceitec.cz
May 05, 2025
Mgr. Milan Esner, Ph.D.
CELLIM, Ceitec MU
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Why Do We Need Labelling in Microscopy?
❗ Native biomolecules are invisible
- Proteins and structures are transparent and colorless under light microscopy.
- Labelling is necessary to detect and analyze them.
🔍 What Labelling Enables:
Ø Visualize the invisible: make specific proteins or structures detectable.
Ø Gain molecular specificity: distinguish between closely related components.
Ø Understand spatial organization: identify compartments and molecule distribution.
Ø Track dynamic processes: observe changes over time (e.g., mitosis, transport).
Ø Study molecular interactions: reveal colocalization and functional associations.
🧪 Common Labelling Techniques:
Ø Immunolabelling (antibody-based)
Ø Fluorescent protein tags (e.g., GFP, CFP…)
Ø Chemical dyes (e.g., DAPI, phalloidin)
Ø In situ hybridization (e.g., FISH)
Biological sample preparation for light
microscopy
Ø Preserve the sample structure
stabilize biological structures (proteins, membranes, organelles) from degradation - internal proteases,
external factors.
Ø Enhances the contrast, visibility
increase the binding sites for dyes and antibodies. Permeabilize the cell membrane for entering antibodies.
Ø Refraction index match - mounting media
Mount to media for long term storage - prevent evaporation, contamination,
Check refractive index of the media to match the objective requirements.
Ø Most common workflow
Fixation, permeabilization, blocking, immunostaining with labelled antibodies, mounting
Biological samples are not stable. They are prone to degradation/contamination over the time. Necessary to fix
the sample to preserve its structure and protect from contamination, before starting the visualization.
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Common used Fixations
Method Advantages Disadvantages Fluorescent proteins
compatibility
Aldehyde based fixation
(Formaldehyde,
Glutaraldehyde)
Preserves structure well,
compatible with most of
immunostainings. Preserve
fluorescence of fluorescent
proteins.
Can cause crosslinking
artifacts after prolonged
period, may alter antigenicity
Yes, but prolonged
fixation can reduce
fluorescence
Alcohol-based Fixation (e.g.,
Methanol, Ethanol)
Rapid dehydration, good for
cytoskeleton preservation.
Can cause cell shrinkage,
may not preserve proteins
well. Loose soluble proteins.
No, GFP fluorescence
is reduced or lost
Acetone Fixation Fast, preserves lipids, good
for some immunostaining
Harsh, can extract proteins No, GFP fluorescence
is lost
Cryofixation (e.g., Liquid
Nitrogen, High-pressure
Freezing)
Best structural preservation,
no chemical artifacts
Requires specialized
equipment, costly
Yes, excellent
preservation. Not for
light microscopy.
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Common used Fixations
Mikroskopicke praktikum IIhttps://blog.cellsignal.com/successful-immunofluorescence-fixation-and-permeabilization
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Formaldehyde CH2O fixation
🔬 **Overview:**
Formaldehyde is a widely used fixative that crosslinks proteins by forming methylene bridges,
preserving cellular structures while maintaining antigenicity. Most common 4% formaldehyde in PBS.
Makes chemical crosslinks between lysine molecules of proteins.
✅ **Advantages:**
- Preserves cellular and tissue morphology effectively.
- Suitable for immunostaining and fluorescence microscopy.
- Maintains protein antigenicity for antibody labeling.
- Compatible with GFP and other fluorescent proteins (short fixation times recommended).
❌ **Disadvantages:**
- Can cause some crosslinking artifacts.
- Penetration into tissues is relatively slow.
- Can increase background fluorescence.
- Requires neutralization or washing to remove unreacted formaldehyde.
- Solution is unstable, often used with methanol as stabilizer.
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Formaldehyde versus glutaraldehyde
Formaldehyde Glutaraldehyde
Chemical Type Monoaldehyde (HCHO) Dialdehyde (OHC–(CH₂)₃–CHO)
Cross-linking Mild, reversible methylene bridges Strong, stable cross-links
Penetration Fast penetration, slow reaction Slower penetration, fast reaction
Antigen Preservation
Good – suitable for
immunostaining
May mask epitopes – limited use
in immunostaining
Ultrastructural Detail Limited preservation
Excellent preservation – ideal for
EM
Fixation Reversibility Partially reversible Irreversible
Typical Use
Light microscopy, IHC,
immunofluorescence
Electron microscopy, structural
preservation
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⚙ **Protocol Recommendations:**
1. Use **4% formaldehyde** in **PBS or culture medium**
(freshly prepared if possible).
2. Fix cells at **room temperature (10–15 min) or 4°C (20–
30 min)**.
3. Wash with PBS after fixation to remove excess fixative.
4. Proceed with permeabilization (if required) before
staining.
💡 **Best Practices:**
- Avoid over-fixation to preserve fluorescence.
- Store stock solution at **-20°C in aliquots** to prevent
polymerization.
Formaldehyde CH2O fixation
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Permeabilization
Definition:
Permeabilization is the process of temporarily or permanently disrupting the cell membrane, allowing
big molecules (e.g., non permeable dyes, drugs, proteins) to pass inside the cell. Together with fixation
are crucial steps for successful staining.
Methods of Permeabilization:
1.Chemical Permeabilization 🧪
1. Use of detergents (e.g., Triton X-100, saponins, Tweens, NP-40 or other detergents)
2. Use organic solvents (e.g. methanol, acetone)
2.Mechanical Permeabilization ⚙
1. Sonication (ultrasound waves)
2. Electroporation (short electrical pulses)
3. Microinjection (direct insertion of substances)
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Permeabilization with detergents
What is detergent-based permeabilization?
Detergents are amphiphilic molecules (having both hydrophilic and hydrophobic parts) that disrupt the lipid
bilayer of cell membranes, making them permeable to small molecules, antibodies, or stains.
How Detergents Permeabilize Cells?
1. Detergents solubilize membrane lipids, disrupting membrane integrity and creating pores.
2. This makes the membrane leaky and allows molecules (e.g., dyes, antibodies, drugs) to enter the cell.
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Types of detergent
🧪 1. Ionic Detergents
•Head group: Carries a net electric charge (positive or
negative).
•Types:
• Anionic (e.g., SDS – sodium dodecyl sulfate):
negatively charged
• Cationic (e.g., CTAB – cetyltrimethylammonium
bromide): positively charged
🔬 Properties:
•Strongly denaturing – disrupt protein–protein and protein–lipid
interactions by breaking both hydrophobic and electrostatic
bonds.
•Break down membranes effectively
•Often used for complete cell lysis and protein denaturation
(e.g., in SDS-PAGE)
•Can disrupt secondary and tertiary protein structures
⚠ Drawbacks:
•Not suitable if you need to preserve native protein function or
antigenicity
🌊 2. Non-Ionic Detergents
•Head group: Uncharged, typically a sugar (e.g., maltoside) or
ethylene oxide chain.
•Examples:
• Triton X-100
• NP-40
• Tween 20, Tween 80
• Digitonin
🔬 Properties:
•Mild – solubilize membranes without fully denaturing proteins
•Maintain protein activity and interactions better
•Often used in:
• Immunostaining
• Immunoprecipitation
• Membrane protein extraction
• Cell permeabilization (e.g., Triton X-100 for nuclear
staining)
✅ Benefits:
•Ideal for applications needing permeabilization without lysis
•Preserve antibody binding sites and epitope structure
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Types of detergent
Key Considerations:
•Higher detergent concentrations or longer incubation times increase permeability but can also damage
cellular structures.
•Cold incubation (4°C) minimizes protein denaturation, while room temperature or 37°C can enhance
permeability.
•Some detergents (like Saponin and Digitonin) provide reversible permeabilization, useful for live-cell
studies.
Detergent Type Typical Concentration Incubation Time Notes
Triton X-100 Non-ionic 0.1%–1% 5–15 min Common for immunostaining, permeabilizes all membranes
Tween-20 Non-ionic 0.05%–0.5% 5–20 min Milder than Triton X-100, used for surface antigens
Saponin Natural 0.01%–0.2% 5–30 min Reversible permeabilization, selective for cholesterol-rich membranes
Digitonin Natural 0.001%–0.02% 5–15 min Selectively permeabilizes plasma membrane but leaves organelles intact
NP-40 Non-ionic 0.1%–1% 5–15 min Stronger than Triton X-100, often used for cell lysis
CHAPS Zwitterionic 0.1%–1% 10–30 min Gentle on membrane proteins, good for maintaining protein activity
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Blocking non specific sites
🎯 What Is Blocking?
Blocking is a preparatory step in immunodetection techniques, used to prevent non-specific binding of
antibodies to the sample. A protein- or polymer-based solution is applied to occupy potential non-specific
binding sites before the addition of the primary antibody.
🔒 Targets of Blocking Include:
•Non-specific membrane surfaces
•Charged functional groups
•Fc receptors, particularly in immune cells
•Hydrophobic regions on proteins or sample supports
🧪 Common Blocking Agents:
•Bovine Serum Albumin (BSA)
•Normal serum (species-matched)
•Fish gelatin
✅ Purpose:
To reduce background signal and enhance the specificity and clarity of antibody-based detection by ensuring
binding occurs only at target epitopes.
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Blocking non specific sites
Blocking Agent Commonly Used In Mechanism
Bovine Serum Albumin (BSA, 1–
5%) Western blot, ELISA, IF Binds to non-specific protein sites
Non-fat Milk (2–5%) Western blot Blocks hydrophobic interactions
Serum (5–10%)
Immunohistochemistry (IHC),
IF Contains proteins to block Fc receptors
Gelatin (0.2–0.5%) Immunostaining Forms a protein barrier on surfaces
Casein (0.5–2%) ELISA, WB Prevents background from hydrophobic sites
Bovine Serum Albumin (BSA) is a globular protein (~66 kDa) derived from cow blood serum.
How BSA Minimizes Non-Specific Binding?
1.Surface Coating 🛡
1. BSA binds to exposed surfaces (e.g., membrane, well plates, slides), preventing antibodies or probes from
sticking non-specifically.
2.Protein Saturation 🚫🔗
1. Many non-specific interactions occur due to empty protein-binding sites on plastic, membranes, or
glass.
2. BSA fills these sites, reducing unwanted background signals.
3.Hydrophobic & Electrostatic Interactions ⚡💧
1. Antibodies and detection reagents can bind non-specifically due to hydrophobic or ionic forces.
2. BSA competes with these interactions, blocking unintended binding.
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Blocking non specific sites
For Immunofluorescence / IHC:
🔹 1–5% BSA in PBS
•Dissolve 1–5 g of BSA in 100 mL PBS
•Optional: Add 5–10% serum from the same species as the secondary antibody.
•Filter with 0.2 um filter
Key Reason: Blocking Fc Receptor Binding
🛡 Immune cells (e.g., macrophages, B cells, dendritic cells) express Fc receptors (FcRs), which can bind antibodies
non-specifically.
🔹 If secondary antibodies bind to Fc receptors instead of their intended targets, this leads to false-positive staining (high
background).
✅ Solution:
•If you use a goat anti-rabbit secondary antibody, block with normal goat serum (NGS).
•If using a donkey anti-mouse secondary, block with normal donkey serum (NDS).
🔬 How It Works:
•The serum contains immunoglobulins (IgG) from that species, which saturate Fc receptors before the secondary antibody
is added.
•This prevents unwanted Fc receptor binding, reducing non-specific fluorescence or staining.
When Is Serum Blocking Important?
✔ Tissue staining (IHC, IF) – Some cells express high Fc receptors, increasing background.
✔ Flow cytometry – FcR-expressing immune cells (e.g., B cells, monocytes) can trap antibodies.
✔ Cell culture assays – Some cell types bind antibodies non-specifically.
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Blocking non specific sites
IgG Antibody – Structure & Function 🧪🔬
What is IgG?
•IgG (Immunoglobulin G) is the most abundant antibody in the blood and extracellular fluid, making up ~75–
80% of total serum immunoglobulins.
•It plays a crucial role in adaptive immunity, neutralizing pathogens and activating immune responses.
IgG Structure 🏗
🔹 Monomeric Y-shaped glycoprotein (~150 kDa)
🔹 Composed of four polypeptide chains:
•2 Heavy Chains (H, ~50 kDa each)
•2 Light Chains (L, ~25 kDa each)
•Disulfide bonds stabilize the structure
Region Function Description
Fab (Fragment antigenbinding)
Antigen recognition Contains variable regions (VH & VL) that bind to specific epitopes
Fc (Fragment crystallizable) Immune activation
Interacts with Fc receptors (FcγR) on immune cells and activates complement
system
Hinge Region Flexibility Allows the antibody to bind two antigens at different angles
Constant Region Effector function Defines IgG subclasses (IgG1, IgG2, IgG3, IgG4) with distinct immune roles
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Common Staining Methods
1. Antibody-based staining
🔹 Primary & secondary antibodies with fluorophores, or labelled primary Abs
🔹 High specificity — targets proteins, structures
🔹 Examples: tubulin, histones, membrane markers
2. Fluorescent chemical compounds
🔹 Small labelled molecules that bind specific cell components cell permeable
🔹 Examples: DAPI / Hoechst → DNA; Phalloidin → actin; MitoTracker → mitochondria
3. Genetically encoded fluorescent tags
🔹 Fluorescent proteins (e.g., GFP, mCherry, mKate, CFP…) fused to target proteins
🔹 Transient or stable expression
🔹 Chromobodies as a subclass (nanobody + FP) – small, cell permeable
4. Fluorescent In Situ Hybridization (FISH)
🔹 Uses fluorescent DNA or RNA probes to detect specific nucleic acid sequences
🔹 Highly sensitive for mRNA, gene loci, or chromosomes
🔹 Often used in diagnostics, gene expression analysis, and cell identification
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Antibody-based staining
O`Kennedy et al., Antibody Technology Journal, 2016
Antigen Primary antibody with fluorophore
Faster, but less sensitive than indirect IF
Fundamentals of light microscopy 19
Direct immunolabelling
Antigen Primary antibody Secondary antibody with fluorophore
Longer protocol, but more sensitive than direct IF due to signal amplification
Fundamentals of light microscopy 20
Indirect immunolabelling
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•Primary antibody binds specifically to the target antigen.
•Secondary antibody, conjugated with a fluorophore, binds to the primary antibody.
•Fluorophore emits fluorescence upon excitation, enabling visualization under a fluorescence
microscope.
✅ Advantages:
•Signal amplification (multiple secondaries per primary).
•Versatile detection using various fluorophores.
•Cost-effective (same secondary used for multiple targets).
Principles of Indirect immunolabelling
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Nanobodies
What are Nanobodies?
•Single-domain antibody fragments derived from camelid antibodies.
•Much smaller than conventional antibodies (~15 kDa vs. ~150 kDa).
•High binding specificity and affinity to target molecules.
Advantages in Microscopy
✅ Better tissue penetration – smaller size allows deeper access.
✅ Higher labeling density – more nanobodies can bind per target.
✅ Minimal background signal – reduced cross-reactivity.
✅ Improved photostability – when conjugated with fluorophores.
Applications in Sample Preparation
🔬 Immunostaining – direct labeling with fluorophore-conjugated nanobodies.
🔬 Super-resolution microscopy – STED, PALM, dSTORM use nanobodies for higher precision.
🔬 Live-cell imaging – minimal perturbation to target proteins.
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Nanobodies
Chromobodies in Light Microscopy
What are Chromobodies?
•Genetically encoded nanoprobes-fluorophore fusion proteins.
•Derived from single-domain camelid antibodies (nanobodies) linked to a fluorescent protein
(e.g., GFP, mCherry).
•Allow real-time intracellular labeling of target proteins.
Advantages in Microscopy
✅ Live-cell imaging – monitor protein dynamics in real-time.
✅ No need for chemical fixation or permeabilization – avoids sample artifacts.
✅ High specificity and minimal background – derived from nanobodies.
✅ Genetically encoded – stable expression in cells.
✅ No permeabilization required - penetrate cell membrane.
Applications
🔬 Tracking protein dynamics – study movement, interactions, and localization.
🔬 Super-resolution microscopy – useful for STED and PALM techniques.
🔬 Functional studies – chromobodies can be fused to reporters for advanced analyses.
🔬 Cell division and cytoskeleton studies – visualize actin, tubulin, and mitotic processes.
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Fluorescent chemical compounds
🔹 **Definition**:
- Small synthetic or natural molecules that bind to specific cellular components
- Provide strong fluorescence signals without genetic modification
🔹 **Key Features**:
- Fast and easy to apply
- Often cell-permeable
- Available in various spectral properties (colors, photostability, pH sensitivity)
- Should not alter other processes or cell viability – not toxic
🔹 **Examples & Targets**:
**DAPI / Hoechst** → DNA (nucleus)
**Phalloidin** → F-actin (cytoskeleton)
**MitoTracker** → mitochondria
**LysoTracker** → lysosomes
**FM dyes** → plasma membrane & endocytosis tracking
✅ Widely used in fixed and live-cell imaging to visualize organelles, structures, and
processes.
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What are Fluorescent Proteins (FPs)?
🔹 Genetically encoded fluorophores
🔹 Fused to a protein of interest → real-time localization
🔹 Expressed in live cells via transfection or stable integration
Common FPs:
•GFP (Green Fluorescent Protein)
•mCherry, RFP, tdTomato (Red variants)
•mTurquoise, CFP (Cyan)
•YFP, Venus (Yellow)
Advantages:
✅ Live-cell compatible
✅ No staining needed
✅ Track dynamic processes (e.g. mitosis, transport)
⚠ Considerations:
•Overexpression artifacts
•Photobleaching
•Requires appropriate filters & stable expression system
Fluorescent proteins
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Fluorescent proteins
• 🔹 **Origin**:
• - First discovered in the jellyfish *Aequorea victoria*
• - Green Fluorescent Protein (GFP) isolated in 1960s, gene cloned in 1992
• 🔹 **Historical Milestones**:
• - 1994: GFP expressed in other organisms → revolution in live-cell imaging
• - 2008: Nobel Prize awarded to Shimomura, Chalfie, and Tsien for GFP
• - Development of color variants: CFP, YFP, mCherry, tdTomato, etc.
• 🔹 **Current Use**:
• - Fused to proteins of interest to monitor localization and dynamics
• - Used in live-cell imaging, biosensors, FRET, photoconversion, and optogenetics
• - Available in multiple colors, photoactivatable or pH-sensitive forms
• ✅ Fluorescent proteins allow real-time, non-invasive visualization of proteins and
processes in living cells.
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Genetically encoded fluorescent tags
Protein Color
Excitation /
Emission (nm)
pKₐ Brightness Photostability Lifetime (ns)
Maturation
Time (h)
Key Features
GFP Green 488 / 509 5.5 ~33 🟢 High 2.6–2.8 ~0.5–1.0
Benchmark FP;
robust & versatile
mNeonGreen Bright Green 506 / 517 5.7 ~90 🟢 High ~3.9 ~0.5
Extremely bright,
fast maturing
YFP Yellow 514 / 527 6.9 ~45 🟠 Medium ~3.2 ~0.7–1.0
FRET donor; pH-
sensitive
CFP Cyan 433 / 475 4.7 ~17 🔴 Low ~2.3 ~1.0
FRET donor; low
brightness
mTagBFP2 Blue 399 / 456 2.7 ~30 🟢 High ~2.7 ~0.6
Bright and
photostable in blue
mCherry Red 587 / 610 4.5 ~16 🟢 High ~1.5 ~1.5
Stable and
monomeric; slower
maturation
tdTomato Orange-Red 554 / 581 4.7 ~95 🟠 Medium ~3.1 ~1.0
Extremely bright,
dimeric
mKate2 Far-Red 588 / 633 5.4 ~34 🟢 High ~3.0 ~0.7
Far-red; great for
deep tissue imaging
Alexa dyes
CF dyes
FITC
Rhodamine
Cy3
.
.
.
https://www.thermofisher.com/cz/en/home/life-science/cell-
analysis/labeling-chemistry/fluorescence-spectraviewer.html
Fundamentals of light microscopy 28
Check he excitation and detection capabilities of the selected microscopy system
Fluorochromes
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What is Photobleaching?
•Loss of fluorescence due to light-induced chemical damage
•Irreversible — leads to fading signal during imaging
Factors affecting photostability:
🔹 Intensity & duration of excitation light
🔹 Fluorophore type (e.g., Alexa Fluor vs. FITC)
🔹 Mounting medium and oxygen levels
How to improve photostability:
✅ Use antifade reagents (e.g., ProLong Gold, Vectashield)
✅ Limit exposure time and excitation intensity
✅ Use photostable dyes (e.g., Alexa Fluor series)
✅ Use fast acquisition modes when possible
Photostability and Fluorophore Protection
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Purpose of Mounting
🔹 Preserves sample for imaging
🔹 Protects against drying and photobleaching
🔹 Refractive index matching for microscopy
Types of Mounting Media
✅ Aqueous media
•Suitable for water-based stains and live imaging
•Example: PBS + glycerol
✅ Hard-setting (curing)
•Long-term storage, coverslip sealing
•Contains antifade reagents to reduce photobleaching
•Example: ProLong Gold, Fluoromount-G
Enable long-term storing of samples
Mounting media and sample preparation
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Step-by-Step Protocol
1⃣ Fixation:
•4% paraformaldehyde in PBS, 10–15 min at RT
2⃣ Permeabilization:
•0.1% Triton X-100 in PBS, 5–10 min
3⃣ Blocking:
•1–5% BSA or serum in PBS, 30-60 min, at RT
4⃣ Primary Antibody Incubation:
•Dilute in blocking buffer, 1 h at RT or overnight at 4 °C
5⃣ Wash:
•3× with PBS, 5 min each
6⃣ Secondary Antibody Incubation:
•Fluorophore-conjugated, 1 h at RT
7⃣ Counterstain (optional):
•e.g., DAPI for nuclei
8⃣ Mounting:
•Antifade medium, seal coverslip
🔬 Ready for fluorescence microscopy
Indirect immunolabelling protocol example
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Live Imaging – SiR-actin Staining
1⃣ Prepare staining solution:
•Dilute SiR-actin (recommended 0.5–1 μM)
•Use phenol red–free, serum-free medium if possible
2⃣ Optional: Add Verapamil
•10 μM to inhibit efflux pumps (improves staining)
3⃣ Incubate cells:
•30–60 min at 37 °C, 5% CO₂
4⃣ Image directly (no wash):
•Live-cell confocal or spinning-disk microscope
✅ SiR-actin is fluorogenic — emits in far-red (exc. 640 nm
/ em. 661 nm)
✅ Compatible with long-term live-cell imaging
Live cell staining protocol example
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Live Imaging – Hoechst + SiR-Actin
1⃣ Prepare staining solution:
•Hoechst 33342: 1 μg/mL
•SiR-actin (Spirochrome): 0.5–1 μM
•Optional: Verapamil 10 μM (improves SiR-actin retention)
•Use phenol red–free, serum-free medium
2⃣ Incubate cells:
•30–60 min at 37 °C, 5% CO₂
3⃣ Image live:
•Hoechst → DAPI channel (exc. 350 nm / em. 461 nm)
•SiR-actin → far-red channel (exc. 640 nm / em. 661 nm)
✅ No fixation or permeabilization needed
✅ Compatible with dynamic, time-lapse imaging
✅ Excellent signal-to-noise and minimal background
Live cell staining protocol example
Dual staining
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What are NIR Fluorochromes?
🔹 Fluorophores excited and emitting in the 700–900 nm range
🔹 Minimal autofluorescence and low light scattering
🔹 Ideal for deep tissue imaging and high-contrast labeling
Examples:
•SiR dyes (e.g., SiR-DNA, SiR-actin)
•IRDye 800CW, Alexa Fluor 750
•Cy7, DyLight 800
Advantages:
✅ Low background fluorescence
✅ Penetrates deeper into tissues
✅ Less phototoxicity — ideal for live imaging
⚠ Considerations:
•Requires specialized filter sets or detectors
•Lower quantum yield than visible-range dyes
•May be less bright on conventional microscopes
Near infrared (NIR) fluorochromes
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Fix and stain cellular structures using an anti-Tubulin antibody, Hoechst, and Phalloidin to visualize the nucleus
and tubulin-actin cytoskeleton. Subsequently, measure the size and morphology of the nuclei and cytoplasm.
Practical part I.
Visualize nucleus and tubulin-actin cytoskeleton
Cells:
Mouse Embryonic Fibroblasts
Reagents:
Hoest 33342 (ThermoFisher, Cat. 62249)
PBS
Triton-X100 (Merck, Cat. T8787)
Tween 20 (Merck, Cat. P9416)
Bovine Serum Albumine (Merck, Cat. A7030)
Anti alpha tubulin, mouse monoclonal (Merck, Cat. T7451)
Anti mouse IgG - Alexa 488 (Thermo, Cat. A11001)
Phalloidin-Alexa647 - (Merck, Cat. 65906)
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Step-by-Step Protocol - ultra fast protocol
1⃣ Fixation: 4% paraformaldehyde in PBS, 10 min at RT
2⃣ Wash: 3× with PBS, 2 min each
3⃣ Permeabilization: 0.5% TritonX100 in PBS, 10 min at RT
4⃣ Blocking: 3%BSA in PBS, 15 min at RT
5⃣ Primary Ab: Add primary Ab, 20 min at RT
6⃣ Wash: 3× with PBS, 2 min each
7⃣ Secondary Ab: Add secondary Ab, 1:1000 dilution,10 ug/ml of Hoechst, Phalloidin 1:500, 20 min at RT
8⃣ Wash: 3× with PBS, 2 min each
9⃣ Mount: remove PBS and add drop of Vectashield (optinally you can add coverslip on top)
🔬 Ready for fluorescence microscopy
Practical part I.
Visualize nucleus and tubulin-actin cytoskeleton
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Practical part I.
Visualize nucleus and actin cytoskeleton in fixed cells
Acquire at least 10 cells
1) measure area, length axis X,Y of nuclei. Calculate mean value.
2) measure thickness of actin and tubulin fibres. Calculate mean value.
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Practical part II.
Stain cells with Hoechst and sir-Act dye in order to visualize nucleus and actin cytoskeleton without
fixation in live cells.
Measure size and morphology of nuclei and cytoplasm in living cells.
Cells: Mouse Embryonic Fibroblasts
Dyes: Hoechst (ThermoFisher, Cat. 62249), sir-Act dye (Spirochem, Cat. SC006)
Step-by-Step Protocol
1⃣ Stain: 10 ug/ml of Hoechst 33342 and sir-Act (1:1000) in cell culture media, 30 min at RT
2⃣ Optional (Wash and add fresh media)
🔬 Ready for fluorescence microscopy
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Practical part II.
Visualize nucleus and actin cytoskeleton in living cells
Acquire at least 10 cells
1) measure area, length axis X,Y of nuclei. Calculate mean value.
2) measure thickness of actin fibres. Calculate mean value.