Sample Preparation Instructions

Preferred Buffer and Fixatives: 
Buffet- 0.1M Sodium Cacodylate 

Fixative for morphological studies – "Epon or Durcapan embedding" 

2 percent paraformaldehyde/ 2-2.5 percent Glutaraldehyde in 0.1M sodium Cacodylate; pH 7.2-7.4

Fixative for immunohistochemical studies- Lowicryl embedding

4 percent paraformaldehyde/0.125 percent Glutaraldehyde in PBS-A; pH 7.2-7.4

Tissue Embedding:  
Tissue needs to sit in a specific fixative for at least a week before processing.  

Fresh tissue is preferred, however we have had success with post mortem tissue (NEVER frozen, unless properly cryoprotected.) We can provide the fixative, and store the samples in our aldehyde storage 4C cold room. 

Cultured Cells Embedding: 
Cells need to sit in fix for at least 24 hours before processing.  

Cells are to be grown on Lab-Tek Permanox Chamber Slides. (These can be purchased through Electron Microscopy Sciences, EMS .Slides come in 1, 2, 4, or 8 chambered slides. Catalog numbers start with #70380). Our facility does not provide the slides.  

 We will provide you with wash buffer and fixative. When the cells are about 75-85 percent confluent, you will 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 TEM facility needs a visible pellet (with the human eye) for the embedding to be successful.  Viruses need to be fixed before we handle the sample.  

1. Plan well before you start. 

Before you start check if you have all the reagents you will need. Prepare all solutions fresh; if using common stocks, make an aliquot for your experiment to ensure you have enough reagents. 

Consider sample preparation and special coating of the coverslip, e.g., cell confluency can affect staining efficiency. 

Choose the best fluorophore/dye for your imaging (see specific protocols for STED/ Multiphoton/ Light-sheet staining). 

Think about additional markers, for instance to visualize specific cellular structures you can use membrane, nuclear, or other stains. 

• Choose the right coverslips.

Ensure that you use correct thickness coverslips. We recommend using #1.5 coverslips (170um thickness) as this may affect the quality and resolution of your images. Ref: The importance of #1.5 thickness coverslips 

• Cell Membrane Staining Vibrant Cell-labeling solution CellBrite Cytoplasmic Membrane Dyes Nile Red (compatible with STED 757nm) FM 4-64 (PI dye)

2. Don’t forget to prepare controls
   When you optimize your protocol and trying different conditions it is very important to prepare controls. Since some of the antibodies may cross-react, controls will help you assess background and unspecific binding. Especially in tissue it is not uncommon to have autofluorescence in the 500-550nm emission range (similar to EGFP, Alexa488); prepare controls to assess autofluorescence. 

You can consider preparing samples: 

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

3. Try different fixation methods.
   Fixation may be harsh and affect your sample structure/morphology or quench the fluorescence of endogenously expressing chromophores. Sometimes it is good to spend time to optimize your fixation method. 

• 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 (1% better than 4%, if possible) for 10-20 mins 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 (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: More gentle with epitopes but also causes removal of lipid components. Similar to methanol, a single step also serves for the permeabilization of cells. Unlike Methanol it is not recommended for preserving cytoskeletal components/structure. 
• Glutaraldehyde: Less used than the previous ones, 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 - 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.

Ref: Fixation artifacts and how to minimize them 

4. 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 will be one containing serum from the same species in which the secondary antibody was raised. In many cases, bovine serum albumin (BSA) at a 5 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. 
5. 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 ones. 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 - MOWIOL is a PVA-based medium originally designed for EM. It's not good 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
     Mowiol Protocol 

6. Prolong Antifade Mountants

 Ref: POSTER- Matching RI of mounting media 

Improving Axial Res in confocal microscopy with new high RI mounting media 

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 (linear unmixing). 

Excitation spectra need to be considered as to avoid cross-excitation. Common culprits are A568 and A594, which both excite with the common 561nm excitation line (you think you are exciting only 568, but you are exciting both 568 AND 594). 

Species cross reactivity for antibodies 
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 have to be absolutely certain that 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. Even better, directly conjugate your antibodies to fluorophores (there are kits for this). 

Secondary Antibodies 
Ensure that your secondary antibodies were produced in a different species than the primary Abs. 
If you used for example Goat, Rabbit, 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 have to do special (sequential) protocols, or use fancy antibodies against specific fragments of your primary Abs. There might also 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, and then use it for the whole panel (e.g. donkey). This simplifies blocking and other protocol steps. (i.e. might enable you to add multiple primary Abs at one step instead of sequentially) 

Please think of adding all immunolabeling controls while validating antibodies. Typical staining controls for a single and a double labeling look like: 

For further info, please refer to these links: 

https://www.jacksonimmuno.com/secondary-antibody-resource/technical-tips/cross-adsorbed-and-cross-reactivity/ 

https://www.jacksonimmuno.com/technical/products/antibody-selection 

https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/9780470089941.et0902s10 

https://bitesizebio.com/44370/controls-for-immunofluorescence-a-beginners-guide/ 

Please confirm with Microscopy CoRE staff about sample prep guidelines and STED compatible dyes

For choosing the right fluorophores and clearing method please ask Microscopy CoRE staff. 

Tissue clearing Webinars from Miltenyi Tissue Clearing Webinar 

Points to note when preparing samples for imaging under multiphoton microscopes: 

1. For simultaneous imaging, DAPI is essentially incompatible because it will bleed-through into all channels. Sequential imaging via sequence manager is the only option if DAPI must be used. 
   • Tune the laser at 750nm for DAPI then 950 nm for AF488 using sequence manager. Notice that there is no bleed over.  
2. AF568 and AF594 cannot be used together on the MPE and really only can be used together on a spectral descanned 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 both 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).  

Here are some examples for the fluorophore combinations.  Fluorescent protein tags tend to excite well at 2P (FPdatabase.org has some 2P data reported for them).  

Simultaneous Multi-color Imaging*  

   Cy3 is technically orange, but the filters should place it at the red channel. Not perfect, but in theory it should excite well at 1045nm  

Multi-color Imaging in Frame Scan mode**  

   ** Far reds and possibly reds may be weak. An SDM565 reverse dichroic sending longer wavelengths to the GaAsP may help.

Helpful Citations:   

1. https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/technical-notes-and-product-highlights/fluorescent-probes-for-two-photon-microscopy.html 
2. https://www.urmc.rochester.edu/research/multiphoton/online-magic-education/fluorophore-two-photon-excitation.aspx 
3. http://www.microscopist.co.uk/general-techniques/fluorophore-databases/ 
4. https://www.drbio.cornell.edu/cross_sections.html 
5. http://www.microscopist.co.uk/wp-content/uploads/2017/04/Excitation-spectra-2P-probes.pdf 
6. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/super-resolution-microscopy/two-photon-microscopy.html 
7. https://www.urmc.rochester.edu/research/multiphoton/online-magic-education/fluorophore-two-photon-excitation.aspx 
8. http://bioemergences.iscpif.fr/bioemergences/tutorials/multiphoton-microscopy.php 
9. https://view.highspot.com/viewer/60a6c60966bbaa41530956f9?iid=60a6c5b9628ba240da98abf8 
10. https://escholarship.org/content/qt9711c98j/qt9711c98j.pdf?t=pdg4gn