Preferred Buffer and Fixatives:

• Buffer- 0.1M Sodium Cacodylate 
• Fixative for morphological studies – "Epon or Durcapan embedding"
• Two percent paraformaldehyde/ 2-2.5 percent Glutaraldehyde in 0.1M sodium Cacodylate; pH 7.2-7.4
• Fixative for immunohistochemical studies-Lowicryl embedding
• Four percent paraformaldehyde/0.125 percent Glutaraldehyde in PBS-A; pH 7.2-7.4

Tissue Embedding:  

• For at least a week before processing, the tissue needs to sit in a specific fixative. Fresh tissue is preferred, however, we have had success with postmortem tissue that is never frozen, unless properly cryoprotected. The CoRE can provide the fixative and store the samples in our aldehyde storage 4C cold room. 

Cultured Cells Embedding:

• For at least 24 hours before processing, cells need to sit in a fixative.
• Our facility does not provide slides. Cells are to be grown on Lab-Tek Permanox Chamber Slides, which can be purchased through Electron Microscopy Sciences, and come in one, two, four, or eight-chambered slides. Catalog numbers start with #70380.
• The CoRE does provide wash buffer and fixative. When the cells are about 75-85 percent confluent, take the cells out of the incubator and wash normally with our sodium cacodylate buffer, then add on the fixative, enough to fully cover the cells. After fixative has been added, we can take the cells back to our lab for storage and dispose of the excess fixative if applicable. 

Suspended cells/cell fractions:

• Our transmission electron facility requires a pellet visible to the human eye for the embedding to be successful. Viruses need to be fixed before we handle the sample.  

Plan well before you start. 

Before beginning, ensure you have all necessary reagents prepared fresh, making aliquots from common stocks if needed. Consider sample preparation factors like cell confluency, which can affect staining efficiency, and determine if special coverslip coating is required. Select the most appropriate fluorophore or dye for your imaging technique, consulting specific protocols for methods like STED, Multiphoton, or Light-sheet staining. Consider incorporating additional markers, such as membrane or nuclear stains, to visualize specific cellular structures. Choose the correct coverslips, paying particular attention to thickness. It's recommended to use #1.5 coverslips with a thickness of 170μm, as this can significantly impact image quality and resolution. Cell membrane staining vibrant cell-labeling solution CellBrite Cytoplasmic Membrane Dyes Nile Red (compatible with STED 757nm) FM 4-64 (PI dye).

Don’t forget to prepare controls.

In optimizing protocol, trying different conditions is critical to preparing controls. Since some of the antibodies may cross-react, controls will help you assess background and unspecific binding. It is not uncommon, especially in tissue, to have autofluorescence in the 500-550nm emission range (similar to EGFP, Alexa488), making it necessary to prepare controls to assess autofluorescence. 

Consider preparing samples with the following: 

• One of each variant in the protocol (different fixation method and antibody concentration)
• Nonspecific-binding control (just primary/just secondary antibody)

Try different fixation methods.

In certain circumstances, it may be helpful to spend time optimizing your fixation method. Fixation may be harsh and affect your sample structure/morphology or quench the fluorescence of endogenously expressing chromophores.

• Paraformaldehyde (PFA): Conserves cellular morphology as it is a chemical cross-linker. It can affect antibody access to some antigens and should be used at low concentrations (one percent better than four percent, if possible) for 10-20 minutes at room temperature. PFA can also denature proteins and affect fluorescence signal and increase background levels. A washing step after fixation with PFA using glycine is sometimes used to quench the PFA and terminate the cross-linking reaction. An additional permeabilization step using detergents (i.e. TritonX, NP-40, Tween, saponin, or others) for cells— for bacteria organic solvents, or detergents with or without lysozyme—is indispensable to allow for access of the antibodies into the sample. Note that gentler permeabilizers (like saponin) may need to be used in every step throughout the staining (antibody incubation, washes) to avoid reversibility. Some ionic permeabilizers (like TritonX) can be used as well in every step at low concentrations to compete non-specific antibody binding. 
• Methanol: Conserves cellular structures but can denature several epitopes, as it dehydrates the sample and is not recommended for state-specific antibodies, like phospho-antibodies. It also compromises lipid integrity, so avoid using when looking at membrane-associated antigens. Using methanol also saves time, as it permeabilizes cells at the same time. It is always used at cold temperatures.
• Acetone: Gentler with epitopes but also causes removal of lipid components. Like methanol, a single step also serves for the permeabilization of cells. Unlike methanol, it is not recommended for preserving cytoskeletal components/structure. 
• Glutaraldehyde: Less frequently used, as chemical cross-linking is even stronger than PFA, which can modify cell or tissue architecture. It is sometimes used in combination with PFA as a mixture for fixing samples. Glutaraldehyde can give rise to strong autofluorescence on tissue that precludes the observation of specific immunofluorescence staining. PFA together with Glutaraldehyde preserve mitochondria morphology better than each alone. See The Combination of Paraformaldehyde and Glutaraldehyde.
• After fixation and washing in PBS you may incubate your cells/tissue with 50mM NH4Cl for few minutes to quench autofluorescence.

For reference: Fixation artifacts and how to minimize them 

Use blocking reagents.

To reduce non-specific antibody binding and, in consequence, reduce background signal, it is recommended to use a blocking solution prior to incubation with antibodies. The most effective blocking solution is one containing serum from the same species in which the secondary antibody was raised. In many cases, bovine serum albumin (BSA) at a five percent dilution will work as a general protein blocker that can be used with any secondary antibody. Blocking reagents can be maintained throughout the staining protocol and added to all solutions, including antibody dilutions, to lower the probability of non-specific binding of antibodies. 

Choose the right mounting medium.

TDE (2,2’-thiodiethanol) nontoxic embedding medium, which, by being miscible with water at any ratio, allows fine adjustment of the average refractive index of the sample ranging from that of water (1.33) to that of immersion oil (1.52). TDE thus enables high resolution imaging deep inside fixed specimens with objective lenses of the highest available aperture angles and has the potential to render glycerol embedding redundant. The refractive index changes due to larger cellular structures, such as nuclei, are largely compensated. Additionally, as an antioxidant, TDE preserves the fluorescence quantum yield of most of the fluorophores. Causes strong GFP quenching. Ref: TDE a new water-soluble mounting medium.

Vectashield, a glycerol-based medium, is one of the most widely used. It can, however, be unsuitable for imaging modes that rely on the red end of the spectrum, as it can generate autofluorescence or be less good at anti-fading. Not recommended with Alexafluor 647. Ref: Effect of Vectashiled-induced fluorescence quenching Mechanism and advancement of antifading agents.

Mowiol is a PVA-based medium originally designed for EM. It's not suitable for 3D imaging. The refractive index is inconsistent between batches. Possible linked to the fact that it arrives as a powder and requires reconstitution in glycerol and buffer. Mowiol has been reported to be involved in PFA-fixation induced redistribution of GFP-tagged transmembrane proteins. See Mowiol Protocol. 

Please confirm with Microscopy CoRE staff about sample prep guidelines and STED compatible dyes. Likewise, to learn how to choose the right fluorophores and clearing method for the lightsheet microscope, please reach out to Microscopy CoRE staff. A tissue clearing webinar from Miltenyi is available, and we have provided additional information, as well.

• Spectral Separation 
  Emission spectra need to be sufficiently separated to be visualized individually. If we expect overlap, we need to do extra work to spectrally unmix them, in a process known as linear unmixing. Excitation spectra also need to be considered as to avoid cross-excitation. Common culprits are A568 and A594, which both excite with the common 561nm excitation line. 
• Species Cross Reactivity for Antibodies
  With primary antibodies, avoid using a primary raised in the same animal species as your tissue (for example anti-mouse for mouse tissue), as you can expect high off-target cross reactivity (background). If you want to go down that route, you must be certain your (usually monoclonal) Ab is very clean.
  For multiplexing, use antibodies raised in different species (or from different Ig classes) for each of your target antigens. Optimally, use a kit to directly conjugate your antibodies to fluorophores.
• Secondary Antibodies
  First, ensure that your secondary antibodies were produced in a different species than the primary Abs. For example, if you used goat, rabbit, or rat primaries on mouse tissue, ensure you are not using secondaries produced in goat, rabbit, rat, or mouse (your target tissue), since such secondaries will also bind to other secondaries in the panel of the species they were raised against. If this cannot be avoided, you must complete special sequential protocols, or use fancy antibodies against specific fragments of your primary Abs. There may be some kits available to address this issue.
  Remember that the secondary will recognize other secondaries if it targets the species the other secondaries were raised in. For example, an anti-goat secondary will recognize any antibodies (primary or secondary) raised in a goat.
  A good strategy is to choose a species for your secondary, then use it for the whole panel (e.g., donkey). This simplifies blocking and other protocol steps and might enable you to add multiple primary Abs at one step instead of sequentially).
  Please think of adding all immunolabeling controls while validating antibodies. We have provided a chart of typical staining controls for a single and a double labeling.

When preparing samples for imaging under multiphoton microscopes, please consider the following: 

• For simultaneous imaging, DAPI is incompatible as it will bleed-through into all channels. Sequential imaging via sequence manager is the only option if DAPI must be used. To achieve this, tune the laser at 750nm for DAPI then 950 nm for AF488 using sequence manager.
• AF568 and AF594 cannot be used together on the MPE and only can be used together on a spectral de-scanned detector microscope. Both fluorophores have similar chemical scaffolds and are likely going to excite simultaneously at nearly the same 2P wavelengths. Once excited, they will both emit red light, and will be detected in the red channel.   

Overall, the user is going to have to select dyes that can be excited simultaneously but with emission peaks that are far enough apart that the filters can separate the emissions into different channels. Then they will need to select dyes that can be excited at different wavelengths from the “simultaneous” batch, preferably utilizing the 1045nm laser if they need simultaneous.