TITLE: Immuno-correlative light and electron microscopy (iCLEM) using STEM
AUTHORS: Viola Oorschot, Jillian  C Danne, Benjamin Lindsey, Jan Kaslin, Georg   Ramm

[DESCRIPTION]
Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy and the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu sample preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for example, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-STEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has diverse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-STEM involves the examination of ultrathin tissue sections (62-70 nm) using immunofluorescence labelling and subsequent SEM imaging to obtain a high resolution overview of these sections with greater morphological detail compared to iCLEM-SEM. This protocol should be of particular interest to EM facilities with SEM, but not TEM access.


[STEPS]
SECTION: Tissue fixation
1. Dissect out the tissue of interest (example, forebrain of Tg(proliferating cell nuclear antigen:GFP) transgenic adult zebrafish with olfactory bulbs attached for tissue orientation) on a teflon plate or dental wax sheet using fine forceps and a scalpel blade, at room temperature and place in fixative, either 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1M phosphate buffer (PB) (pH 7.4) or 4% paraformaldehyde in 0.1M PB (pH 7.4). Keep the tissue submerged in fixative at all times.

Always perform fixation using a fume hood and wear appropriate personal protective equipment (PPE). 
SECTION: Tissue fixation
2. Place the tissue in 5 ml tubes containing the fixative used in Step 1 (2% paraformaldehyde, 0.2% glutaraldehyde in 0.1M PB (pH 7.4) or 4% paraformaldehyde in 0.1M PB (pH 7.4)) and fix overnight, at 4 degrees Celsius on a rotor. For brain samples, add 4% sucrose to the fixation solution.
SECTION: Tokuyasu embedding and sectioning
4. Remove the fixative and wash with phosphate buffered saline (PBS), 3 x 10 mins.
SECTION: Tokuyasu embedding and sectioning
5. Wash with 0.15% glycine in PBS for 10 mins. 
SECTION: Tokuyasu embedding and sectioning
6. Infuse tissue with pre-warmed 6% gelatin in 0.1M PB for 1 hour at 37 degrees Celsius, agitating. Use a heat block to maintain the temperature.
SECTION: Tokuyasu embedding and sectioning
7. Infuse tissue again with pre-warmed 6% gelatin in 0.1M PB for 1 hour at 37 degrees Celsius, agitating.
SECTION: Tokuyasu embedding and sectioning
8. Place gelatin infused tissue in plastic embedding moulds containing pre-warmed 6% gelatin in 0.1M PB at 37 degrees Celsius, and solidify gelatin blocks by cooling at 4 degrees Celsius for 60 mins.

Alternatively, the flat embedding method can be used to minimise gelatin block shrinkage following sucrose infiltration. Add 6 percent pre-warmed gelatin in 0.1M PB to a petri dish and solidify at 4 degrees Celsius for 1 hour. Place the tissue on top in a layer of 6 percent gelatin pre-warmed to 37 degrees Celsius. Solidify gelatin at 4 degrees Celsius for 1 hour.
SECTION: Tokuyasu embedding and sectioning
9. Place the gelatin infused tissue blocks on a teflon plate or dental wax sheet and cut off excess gelatin from around the tissue using a razor blade or scalpel. 

For flat embedded samples, cut around the tissue using a razor blade or scalpel and remove the gelatin embedded sample from the petri dish using a small spatula. Cut off excess gelatin using a razor blade or scalpel. 
SECTION: Tokuyasu embedding and sectioning
10. Fix the gelatin infused tissue blocks in 0.2% paraformaldehyde in 0.1M PB for 30 mins at 4 degrees Celsius, rotating.
SECTION: Tokuyasu embedding and sectioning
11. Wash tissue blocks with 0.1M PB, 3 x 10 mins, agitating.
SECTION: Tokuyasu embedding and sectioning
12. Infuse tissue blocks with 2.3M sucrose in 0.1M PB for 2 days at 4 degrees Celsius, rotating.
SECTION: Tokuyasu embedding and sectioning
13. Mount each block on a clean aluminium bullseye pin. Position tissue in the correct orientation and remove the excess sucrose with strips of filter paper. 
SECTION: Tokuyasu embedding and sectioning
14. Freeze each sample block by gently submerging the pins in liquid nitrogen. For large blocks, cool the sample pin in the chamber of a cryo-ultramicrotome (-100 degrees Celsius) for 10 mins prior to freezing in liquid nitrogen. Pins should be mounted and frozen one at a time. 
SECTION: Tokuyasu embedding and sectioning
15. Trim the front face and edges of the frozen tissue block cutting 50-100 μm deep on all four sides. Cut at a speed of 100 mm/sec with a 100 nm feed, at -90 to -100 degrees Celsius using a Leica UC7/FC7 cryo-ultramicrotome and Diatome cryotrim 20 knife.
SECTION: Tokuyasu embedding and sectioning
16. To check the region of interest:
SECTION: Tokuyasu embedding and sectioning
16.1. Cut semi thin sections with a feed of 100-300 nm, at 3 mm/sec and at -90 to -100 degrees Celsius. 
SECTION: Tokuyasu embedding and sectioning
16.2. Pick up sections in a 1:1 mixture of 2% methylcellulose: 2.3M sucrose in 0.1M PB using a 3 mm stainless steel loop.
SECTION: Tokuyasu embedding and sectioning
16.3. Place sections on a slide and stain with methylene blue/Azure II or toluidine blue solution for 10-20 seconds before rinsing with water and drying on a mini hotplate.
SECTION: Tokuyasu embedding and sectioning
16.4. Observe sections with a wide-field light microscope. If the region of interest has not been obtained, continue trimming and repeat Step 16.
SECTION: Tokuyasu embedding and sectioning
17. Once the region of interest has been obtained, cut 62-70 nm ultrathin sections at 0.8 mm/sec and -90 to -100 degrees Celsius using a Leica UC7/FC7 cryo-ultramicrotome and Diatome cryo immuno knife.
SECTION: Tokuyasu embedding and sectioning
18. Pick up ultrathin sections in a 1:1 mixture of 2% methylcellulose : 2.3M sucrose in 0.1M PB using a 3 mm stainless steel loop, and place sections on 50-150 mesh copper or palladium coated copper grids with carbon-coated formvar film.

Store grids in an enclosed petri dish at 4 degrees Celsius until use.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
20. 
Fill the wells of a 24-well plate with PBS and float grids section side down.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
21. Use a fine tip pasteur pipette to remove enough PBS from each well so that the lid can be secured on the well-plate without disturbing the floating grids. Seal the lid with parafilm.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
22. Put the well-plate containing the grids in an oven set to 50-60 degrees Celsius for 1 hour to remove the 2% methylcellulose : 2.3M sucrose and 6% gelatin from the tissue sections.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
23. Place a sheet of parafilm on a clean flat bench or a glass board using a small amount of water underneath to keep the film flat. 

For the following steps, use a perfect loop or fine forceps to transfer grids from one drop of solution to another, section side down. Use approximately 100-200 μl drops for rinsing solutions and 5 μl drops for antibodies on a clear parafilm surface. Centrifuge all antibodies for 30 seconds using a benchtop microcentrifuge prior to use.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
24. Quench aldehydes with 0.15% glycine in PBS, 5 x 2 mins.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
25. Block with 1% bovine serum albumin (BSA) in PBS for 5 mins. 
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
26. Incubate with both primary antibodies diluted in 1% BSA/PBS for 45 to 60 mins in a dark moist chamber, at room temperature. 

Example: Mouse anti-glutamine synthetase (1:500 dilution), to label glial cells. 

Example: Biotinylated anti-eGFP (1:300 dilution), to label green fluorescent protein labelled proliferating cell nuclear antigen (PCNA).
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
29. Rinse with 0.1% BSA in PBS, 5 x 2 mins.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
30. Incubate with both secondary antibodies diluted in 1% BSA/PBS for 45 mins in a dark moist chamber, at room temperature. 

Example: Goat anti-mouse AlexaFluor-555 (1:300 dilution).

Example: Goat anti- rabbit AlexaFluor-488 (1:300 dilution)


SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
31. Rinse with PBS, 5 x 2 mins.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
32. Rinse with distilled water, 4 x 2 mins.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
33. Incubate with Hoechst nuclear stain (1μM in distilled water) for 20 mins. 
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
34. Rinse with water, 4 x 2 mins.
SECTION: Fluorescent optical microscopy imaging for grids
35. Place grid section side up in a drop of 50% glycerol in water. Ensure the grid is completely covered in glycerol with no air bubbles present.
SECTION: Fluorescent optical microscopy imaging for grids
36. Place grid section side down on a 30 mm glass bottom culture dish and cover grid with a glass coverslip.
SECTION: Fluorescent optical microscopy imaging for grids
37. Acquire a fluorescent montaged z-stack of 62-70 nm ultrathin tissue sections using an inverted widefield fluorescent microscope.

Example: Leica AF6000LX with a DFC 350FX camera, and a 40x 0.6 NA dry or 63x 1.3 NA glycerol objective. 

After imaging, prepare the grids for scanning electron microscopy as follows:

SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
38. Add distilled water to the 30 mm glass bottom culture dish and agitate gently until the coverslip floats.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
39. Remove the coverslip and lift the grid out using fine forceps.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
40. Carefully remove water from the backside of the grid by placing filter paper at the grid edge. 
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
41. Put a drop of distilled water on a sheet of parafilm and place the grid on the drop. The grid should float.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
42. Rinse with distilled water, 6 x 2 mins. Water drops should be placed on a clean parafilm sheet.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
43. Stain the grid in a drop of 2% Uranyloxalate (pH 7.0) for 5 mins at room temperature. 
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
44. Rinse once with distilled water. 
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
45. Cover a glass petri dish with parafilm using a small amount of water under the film to keep it flat. Place the dish on ice and add 3 drops of filtered 2% methylcellulose : 4% uranyl acetate (9 ml : 1 ml) (pH 4.0) to the surface. 
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
46. Float the grid briefly in the first two drops of methylcellulose/uranyl acetate, then transfer to the third drop and leave for 10 minutes.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
47. Remove and dry the grid following the looping out method:
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
47.1. Clean a remanium wire loop with water and dry.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
47.2. Push the loop into the the methylcellulose/uranyl acetate drop and under the grid.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
47.3. Lift the grid out from the drop using the loop.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
47.4. Tilt the loop and grid at a 45-60 degree angle and bring the loop to a piece of filter paper. 
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
47.5. As soon as the loop contacts the filter paper, drag it slowly along the filter paper to remove excess liquid. A thin film of methylcellulose-uranyl acetate should remain on the surface of the grid. The slower the drag, the thinner the film.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
47.6. Dry the grid in the loop for 30 minutes at room temperature.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
47.7. Remove the grid from the loop using fine forceps, taking care not to tear the methylcellulose/uranyl acetate film away from the grid. Store the grid in a grid box.
SECTION: Preparation of immunofluorescence grids for scanning transmission electron microscopy
48. Place the grid in a specialised STEM grid holder for image acquisition of a high resolution overview using a scanning electron microscope. 

Example: Thermofisher/FEI Nova NanoSEM 450 equipped with a bottom mount STEM II (HAADF) detector set at 30 keV with a working distance of 6.8 mm, dwell time of 3 μs, and with MAPS 2.0 software used for generating montages and correlation.
SECTION: Tissue fixation
3. Samples can be stored in 1% paraformaldehyde in 0.1M PB at 4 degrees Celsius until further processing.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
27. Rinse with 0.1% BSA in PBS, 5 x 2 mins.
SECTION: Immunofluorescent labelling of ultrathin sections for optical microscopy using two cellular markers
28. Optionally incubate with a bridging antibody diluted in 1% BSA/PBS for 30 mins at room temperature. 

Example: Rabbit anti-biotin (1:10,000 dilution) 
SECTION: Tokuyasu embedding and sectioning
19. 62-70nm ultrathin sections can be used for immunofluorescence (IF) labelling for optical microscopy using different cellular markers (Steps 20-37) and then subsequent SEM imaging to obtain a high resolution overview (Steps 38-48).