Sample preparation

Introduction to sample preparation

The microscope is not magical; without a good sample, you won't have images that are suitable for image analysis, but a good design of your experiment could save you a lot of time and money.

1. Choice of the coverslip

In general, you should always use the #1.5 coverslip (0.17mm thickness) as most microscope objectives are designed to work optimally with these. This is true for fixed samples as well as live samples. For live-cell imaging, Ibidi is a very good choice of dishes and chambers.

2. Choice of your fluorophore(s)

First, make you have the right laser lines and/or excitation-emission filters available to match the fluorophores you chose, either in your lab or in the facilities around you.
Second, especially if you are working with a living sample, try to work with fluorophores with a peak in excitation that is toward the longer wavelengths. Shorter wavelengths, such as UV, have stronger energy and will damage your sample faster.
Third, obviously, you want a bright fluorophore (high quantum yield) with a long lifetime. To choose from the myriad of fluorophores, a fluorescent protein visualization tool developed by Talley Lambert will help.
Finally, avoid as much as possible any cross-talk between your fluorophores; to check for that, Fluorescence SpectraViewer from Thermo Fisher Scientific is one option.

Choosing your antibody: BenchSci developed a very useful database of Ab, which can help you in your choice.

3. Fixation and permeabilization

Fixing and permeabilizing your cells affects the cell morphology, protein localization and the availability of the antigen you are trying to detect. You may get different results with different reagents, times and concentrations hence the need for protocol optimization. The distortion of the cell morphology is something to bear in mind when interpreting the images.

Fixation

Formaldehyde/paraformaldehyde: Aldehyde fixatives cross-link proteins and generally preserve well the cell morphology. They are somewhat slower acting than organic solvents, especially for thick specimens. 4% formaldehyde for 10 min is a good starting point for mammalian cells.

(Glutaraldehyde preserves the cellular structure very well but causes strong autofluorescence and so is used mainly for EM.)

Methanol Organic solvents like methanol precipitate proteins, which produces a similar outcome to crosslinking with aldehydes — the protein "shells" of the cell are maintained, but all the small molecules within will be lost during the rest of the protocol. Fixation with methanol denatures proteins, so never use it if you have a fluorescent protein that you want to look at in your sample. Some antigenic epitopes are, in fact, completely destroyed by methanol. However, it might also be advantageous for some other antibodies (especially some monoclonal antibodies which bind only one epitope) that the naturally buried antigens could be exposed to after methanol fixation. Methanol/Acetone Using a combination of methanol and acetone can improve immunolabelling lost in 100% methanol fixation (methanol is best for a structure such as microtubules, acetone permeabilize well and is less damaging). Try a few minutes in a solvent at -20°C as a starting point.

Permeabilization

Permeabilization helps get the antibodies into your now-fixed cells. A range of different detergents can be used, including NP-40, Triton X-100, Tween-20 and Saponin. Other solvents (eg, ethanol or DMSO) can also be used to remove lipid. The fixation step actually permeabilizes the cells to some degree (ie they remove some of the membranes), so these steps aren't really completely distinct (eg Acetone permeabilizes as well as fixes).

The extent of permeabilization required depends on what you are trying to look at. For example, cell surface proteins don't require much/any permeabilization; indeed, if you remove all the membranes, you will lose your protein. Labeling before permeabilization can also help distinguish extracellular subjects (eg, cell surface-bound bacteria) and that within the cells (which only stain after permeabilization).

Staining will be improved by blocking the non-specific binding of your antibody to your specimen. BSA, milk or serum can be used for this. Don't use serum from the same species as the one in which the primary antibody was raised!
If one uses a secondary antibody developed from goat, a typical blocking solution is 5% normal goat serum made in PBS (some people also add 0.05% Tween-20 to the solution). NaN3 can be added to 0.05% (w/v) to the solution. Blocking can last from 10 minutes to 30 minutes.

4. Mounting and Recording medium

Mounting medium

Mounting medium helps preserve your sample and raises the refractive index to give good performance with oil objectives. Mountants often have scavengers that soak-up free radicals and reduce photobleaching (these sometimes reduce the initial brightness of your samples).

A glycerol-PBS mixture is suitable for most fixed immunofluorescence and GFP-labeled samples, using from 50-90% glycerol, 1x final PBS, and pH 8-9. An antifading additive is highly recommended. Time and the act of observation both accelerate the loss of fluorescence from any sample. This inevitable process can be slowed by adding various reagents, chiefly antioxidants and radical scavengers, to the mounting medium.

Commercial products are available, such as (in no particular order):

Fluoromount-G (Electron Microscopy Sciences): phosphate-buffered glycerol + 10% polyvinyl alcohol + 0.1% sodium azide.
Aqua/Poly-Mount (Polysciences)
ProLong, SlowFade, SlowFade Light (Molecular Probes):
Vectashield (Vector Laboratories)
Immumount (Shandon)

Here is an illustration of the effect of mounting medium on the structure of the cell:

Recording medium

This really depends on your cells.

Avoid imaging with red phenol as it will add a great amount of background.
If you are having trouble problems with cell death or damage during fluorescence imaging, several commercial products are available to minimize phototoxicity:

Clearing methods

For thick, fixed specimens, consider using clearing methods which will reduce light scattering and the opaqueness of the sample. Here is a table copied from the Nature Methods blog post "Methods and probes for cleared tissue: an imperfect table":

**An imperfect table about some labels that have been used in cleared tissue**
Probe	Labeling shown in large samples^b	Labeling shown in small samples
Genetically encoded fluorescent proteins	3DISCO (signal quenched after a few days), CLARITY, CUBIC, ExM (variant)^a, PACT/PARS (signal retained 6 months and longer)^a, sDISCO^a	BABB (signal quenched after a few hours), ClearT2, ExM (variant)^a , ExM/ePACT^a , ScaleA2, ScaleS, SeeDB, Sucrose, TDE
Immunolabels	3DISCO, CLARITY, iDISCO, iDISCO+^a, iSeeDB, PACT/PARS, Sucrose, SWITCH	BABB, ClearT, ClearT2, CUBIC, SeeDB, SeeDB2^a (in press) Sucrose, TDE, ExM
Specific nucleic acid detection	EDC-CLARITY	CLARITY, ExM (variant)^a, PACT/PARS
Dyes and stains
Congo Red	3DISCO
Lipophilic dyes such as DiI, Sudan Black	PACT/PARS (Sudan Black), SWITCH (DiI)	ClearT, ScaleS, SeeDB
Nuclear stains such as DAPI, DRAQ5, SYTO and PI	CUBIC, ExM (variant)^a, PACT/PARS, SWITCH	SeeDB, SeeDB2^a
SNAP-tags with SiR probes	PACT/PARS, BABB, Scale	SeeDB

Clearing method: Solvent-based; simple immersion; hyperhydration; hydrogel-based.

^aUnpublished information

^bIndicates larger samples such as whole organs

Sources: E. Boyden, MIT; K. Chung, MIT, K. Deisseroth, Stanford University; H-U. Dodt, Vienna University of Technology/Medical University of Vienna; V. Gradinaru, Caltech; P. Heppenstall, EMBL; Takeshi Imai, RIKEN; K. Johnsson, EPFL; J. Lichtman, D. Richardson, Harvard University; A. Miyawaki, RIKEN; M. Tessier-Lavigne, N. Renier, Rockefeller University.

Glossary of some tissue clearing agent acronyms

3DISCO: Three-dimensional imaging of solvent-cleared organs

sDISCO: Stabilized three-dimensional imaging of solvent-cleared organs

BABB: Benzyl alcohol and benzyl-benzoate

CLARITY: Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel

CUBIC: Clear unobstructed brain imaging cocktails and computational analysis

EDC-CLARITY: 1-Ethyl-3-3-dimethyl-aminopropyl carbodiimide-CLARITY

ePACT: PACT-based expansion clearing.

iDISCO: Immunolabeling-enabled 3D imaging of solvent-cleared organs

iDISCO+: Immunolabeling-enabled 3D imaging of solvent-cleared organs plus

PACT: Passive Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel

PARS: Perfusion-assisted agent release in situ

seed : See Deep Brain

Spalteholz’s preparation : Benzylbanzoate/methylsalicate

TDE : 2,2′-thiodiethanol