TITLE: Immuno-correlative light and electron microscopy (iCLEM) using SEM
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 with 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-SEM 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-SEM provides a large quantifiable overview of 200 nm tissue sections without the presence of grid bars, and thicker sections enhance the immunofluorescent labelling.





[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 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 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 200 nm semithin sections at 3 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 semithin 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 directly in a carbon-coated μ-slide 8 well high microscope Ibidi chamber with a #1.5 polymer coverslip surface.

Store sections in a sealed microscope Ibidi chamber at 4 degrees Celsius until use.
SECTION: Tokuyasu embedding and sectioning
19. 200 nm semi thin sections can be used for immunofluorescence (IF) labelling for optical microscopy using different cellular markers (Steps 20-33) and then subsequent SEM imaging to obtain a quantifiable overview of a region of interest (Steps 34-49).
SECTION: Immunofluorescent labelling for optical microscopy using two cellular markers
22. Quench aldehydes with 0.15% glycine in PBS, 5 x 2 mins.
SECTION: Immunofluorescent labelling for optical microscopy using two cellular markers
23. Block with 1% bovine serum albumin (BSA) in PBS for 5 mins. 
SECTION: Immunofluorescent labelling for optical microscopy using two cellular markers
24. 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 for optical microscopy using two cellular markers
25. Rinse with 0.1% BSA in PBS, 5 x 2 mins.
SECTION: Immunofluorescent labelling for optical microscopy using two cellular markers
30. Rinse with distilled water, 4 x 2 mins.
SECTION: Immunofluorescent labelling for optical microscopy using two cellular markers
31. Incubate with Hoechst nuclear stain (1μM in distilled water) for 20 mins. 
SECTION: Immunofluorescent labelling for optical microscopy using two cellular markers
32. Rinse with water, 4 x 2 mins.
SECTION: Fluorescent optical microscopy imaging for ibidi chambers
33. Acquire a fluorescent montaged z-stack of 200 nm semithin sections in distilled water using an inverted widefield 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 semithin sections for scanning electron microscopy as follows:
SECTION: Preparation of sections for scanning electron microscopy
34. Perform the following steps directly in Ibidi chambers and in the fume hood using the appropriate PPE:

Fix 200 nm sections with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4) overnight at 4 degrees Celsius.
SECTION: Preparation of sections for scanning electron microscopy
35. Rinse with 0.1M sodium cacodylate buffer (pH 7.4), 3x 5 mins at room temperature.
SECTION: Preparation of sections for scanning electron microscopy
36.  Post fix with 2% osmium tetroxide and 1.5% potassium ferricyanide in 0.1M sodium cacodylate buffer for 90 mins at 4 degrees Celsius. 
SECTION: Preparation of sections for scanning electron microscopy
37. Rinse with 0.1M sodium cacodylate buffer, 6 x 5 mins at room temperature.
SECTION: Preparation of sections for scanning electron microscopy
38. Fix with 1% Tannic acid in 0.1M sodium cacodylate buffer for 30 mins at room temperature.
SECTION: Preparation of sections for scanning electron microscopy
44. Rinse once with distilled water.
SECTION: Preparation of sections for scanning 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 sections for scanning electron microscopy
46. Float the coverslip with section briefly in the first two drops of methylcellulose/uranyl acetate, then transfer to the third drop and leave for 10 minutes.
SECTION: Preparation of sections for scanning electron microscopy
47. Remove and dry the section following the looping out method:
SECTION: Preparation of sections for scanning electron microscopy
47.1. Clean a remanium wire loop with water and dry.
SECTION: Preparation of sections for scanning electron microscopy
47.2. Push the loop into the the methylcellulose/uranyl acetate drop and under the coverslip/section.
SECTION: Preparation of sections for scanning electron microscopy
47.3. Lift the coverslip/section out from the drop using the loop.
SECTION: Preparation of sections for scanning 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 sections for scanning 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 section. The slower the drag, the thinner the film.
SECTION: Preparation of sections for scanning electron microscopy
47.6. Dry the section in the loop for 30 minutes at room temperature.
SECTION: Preparation of sections for scanning electron microscopy
47.7. Remove the coverslip/section from the loop using fine forceps, taking care not to tear the methylcellulose/uranyl acetate film away from the section. Mount the coverslip/section on an SEM stub using a carbon tab section side up.
SECTION: Immunofluorescent labelling for optical microscopy using two cellular markers
20. Perform the following steps directly in the wells of an Ibidi chamber. Use approximately 300 μl for rinsing solutions and 20 μl for antibodies. Centrifuge all antibodies for 30 seconds using a benchtop microcentrifuge prior to use.


Wash with PBS and place the ibidi chamber at 50-60 degrees Celsius for 1 hour to remove the 2% methylcellulose : 2.3M sucrose and 6% gelatin from the tissue sections. 
SECTION: Preparation of sections for scanning electron microscopy
39. Rinse with 0.1M sodium cacodylate buffer, 6 x 5 mins.
SECTION: Preparation of sections for scanning electron microscopy
40. Incubate sections in 1% osmium tetroxide in distilled water for 30 mins at 4 degrees Celsius.
SECTION: Preparation of sections for scanning electron microscopy
41. Rinse with distilled water, 6 x 5 mins.
SECTION: Preparation of sections for scanning electron microscopy
42. Punch out the tissue section with the polymer coverslip from the Ibidi chamber using a rapid-core 3.5 mm biopsy punch. Handle the polymer coverslip with the tissue section using tweezers or a perfect loop as an EM grid would be handled.
SECTION: Preparation of sections for scanning electron microscopy
43. Transfer the section to a drop of 2% Uranyloxalate (pH 7.0), and stain for 5 mins at room temperature.
SECTION: Preparation of sections for scanning electron microscopy
48. Coat the stub and mounted section with 2 nm of iridium or platinum. 
SECTION: Preparation of sections for scanning electron microscopy
49. Mount the stub on a standard SEM holder for imaging using a scanning electron microscope.

Example: Thermofisher/FEI Nova NanoSEM 450 equipped with a retractable back scatter electron detector set at 10 keV with a working distance of 5 mm, dwell time of 3 μs, and with MAPS 2.0 software used for generating montages and correlation.   
SECTION: Immunofluorescent labelling for optical microscopy using two cellular markers
21. Wash with PBS, 5 x 2 mins at room temperature.
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 for optical microscopy using two cellular markers
26. 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: Immunofluorescent labelling for optical microscopy using two cellular markers
27. Rinse with 0.1% BSA in PBS, 5 x 2 mins.
SECTION: Immunofluorescent labelling for optical microscopy using two cellular markers
28. 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 for optical microscopy using two cellular markers
29. Rinse with PBS, 5 x 2 mins.