TITLE: Correlative Microscopy for Localization of Proteins In Situ: Pre-embedding Immuno-Electron Microscopy Using FluoroNanogold, Gold Enhancement, and Low- Temperature Resin
AUTHORS: Daniela  Boassa

[DESCRIPTION]
Immuno-electron microscopy (immuno-EM) is a technique that has been used widely to determine sub cellular localization of proteins. Different approaches are available for immuno-EM: pre-embedding method, post-embedding, and cryosectioning (Tokuyasu “style”). Here we describe a pre-embedding technique that allows the labeling of a target protein in situ , retention of fluorescence signal in plastic, and its localization at the EM level in a given cellular context. The procedure can be technically challenging and labor intensive: it requires optimization of fixation protocols to better preserve the cellular morphology and screening of compatible antibodies. Nevertheless, immuno-EM can be a powerful localization tool.

[BEFORE_START]
Perform a fixation series of paraformaldehyde and glutaraldehyde dilutions, and assess where the fluorescence signal is lost.

[GUIDELINES]
1. For the localization of intracellular proteins such as Cx43, this protocol necessitates the use of a permeabilizing agent. In general, the use of detergents, although useful to facilitate the penetration of antibodies into cells, is detrimental for the preservation of the overall ultrastructure, which is why we recommend performing the permeabilization step at 4 °C. For Triton X-100 we recommend not to exceed a concentrAtion of 0.1 %. Several types of detergents are available and can be used as alternative to Triton X-100. For example, saponin is gentler with the membranes and is reversible and can be
used to increase membrane permeabilization. Digitonin is another alternative. We recommend testing a series of dilutions with 0.1 % being the highest to assess the lowest concentration of detergent that will achieve a good labeling.

2. The optimal primary antibody dilution should be determined empirically for each antibody used. Also, the primary antibody incubation time can be different depending on the antibody used. Some might require a longer incubation time at 4 °C; others might work well only at room temperature. Whenever possible we recommend working at 4 °C in order to better preserve the ultrastructure.

3. The quenching step with glycine is important to inactivate unreacted aldehyde groups, which may be present after glutaraldehyde fixation. It does not affect negatively the ultrastructure preservation.

4. The size of the final gold particles depends of the time of application. Based on the target of interest the experimenter should adjust the time of gold enhancement.

[STEPS]
1. Remove the culture medium from the MatTek dishes contain ing the cells and wash three times with HBSS pre-warmed up at 37 °C. Fix cells with pre-warmed fixative at 37 °C (4 % PFA, 4 % PFA + 0.1 % glut, 2 % PFA, 2 % PFA + 0.1 % glut) for 5 min at room temperature followed by 30 min over ice. All solutions and steps from this point on are utilized and performed at 4 °C for the preservation of the ultrastructure (see Safety Warnings).
2. Remove fixative and wash cells three times for 2 min each in ice-cold 1× PBS. Note: during any washing step the cells should never become dry to avoid irreversible damage to morphology.
3. Incubate in blocking buffer for 1 h at 4 °C (see Guideline 1).
4. Incubate in primary antibody diluted in blocking buffer at 4 °C for 1 h on a rocking platform set for gentle agitation (see Guideline 2).
5. Rinse cells with Working Buffer, six times for 2 min each at 4 °C.
6. Incubate in secondary antibody diluted in blocking buffer at 4 °C for 1 h on a gentle rocker, protected from light. Remember: keep cells in the dark from now on, to preserve fluorescence.
7. Rinse cells with Working Buffer, six times for 3 min each at 4 °C.
8. Wash cells with ice-cold 1× PBS, three times for 3 min each at 4 °C.
9. Check staining with a fluorescence microscope; proceed with EM processing only if good signal and specificity is observed. An example of the fluorescence immunolabeling for Cx43 in NRK cells is shown below.
  

10. Postfix with 2 % glutaraldehyde in PBS for 10 min at 4 °C.
11. Remove fi xative and rinse three times for 2 min each in ice-cold 1× PBS. Then wash cells for 5 min in quenching solution at 4 °C to remove aldehydes (see Guideline 3).
12. Rinse three times for 5 min each in ddH2O at 4 °C.
13. Gold enhancement: incubate cells in the Gold Enhancement kit for 1–5 min to intensify gold particles. The reaction is light insensitive so it can be carried out under normal room lighting. 

The kit is composed of 4 components: the enhancer (A),the activator (B), the initiator (C), and the buffer (D).

Equilibrate the solutions at room temperature. Right before use, combine equal volumes of A and B first, wait 5 min then add equal amounts of C and D (see Guideline 4).
14. Thoroughly rinse three times for 5 min each in ddH2O at 4 °C.
15. Dehydration (ethanol series): dehydrate in increasing concentration of ethanol: 20, 50, 70, 90, and 2× 100 % ice-cold ethanol for 2 min each. Then rinse with 100 % ethanol at room temperature for 2 min.
16. Infiltration and embedding:
- Remove 100 % ethanol and replace with a 50:50 mixture of low-temperature resin (LR White): ethanol 100 % for 20 min at room temperature.

- Remove the mixture and replace with 100 % LR White for 1 h. MatTek dish can be stored overnight in unpolymerized resin at 4 °C.

-The following day allow three more changes in 100 % of LR White within an hour.
17. Cold-cure procedure: In a glass vial add 10 ml of LR White and one drop of accelerator. Mix well but avoid introducing any air to the solution and immediately add to cells. Cover with a piece of Aclar Embedding fi lm, avoiding any air bubbles. Place MatTek dish inside an aluminum dish with ethanol on ice to keep the temperature low. Keep it on ice until the resin polymerizes (30 min to an hour).

Store at 4 °C.
17.1. At this point the cells can be imaged at the LM level: the signal of the fluorescent label is retained after the embedding procedure and the enhanced gold particles can be viewed by transmitted light. An example is shown in below.
  

18. Sectioning: choose a region containing labeled cells and mount and trim the LR-White block. Cut ultrathin sections (70–90 nm) and collect them on EM copper grids. Dry grids on filter paper and store them in a grid case at room temperature. Examine the ultrathin sections by TEM (see Fig 3).