Localization of proteins in context of the cell's ultrastructure has been a powerful research tool for decades. With this protocol, we extended the proteins that are low in abundance or difficult to locate. We use fluorescence microscopy to label proteins in a sensitive and screenable manner and combine it with electron microscopy to refill the cell's ultrastructure.
Here we will demonstrate on-section CLEM on autophagy proteins, but it can really be applied to any biological question where the cell's ultrastructure is of interest, especially in the study of rare events. Demonstrating the procedure with me will be Tineke Veenendaal, a senior research technician from our laboratory. To begin, replace the PBS containing 0.15%glycine with 1%gelatin in PBS prewarmed to 37 degrees Celsius.
Scrape and transfer the cells in gelatin to a microcentrifuge tube. Pellet the cells at 6, 000 G for one minute at room temperature in a microcentrifuge and then remove the gelatin without disturbing the pellet. Add 12%gelatin warmed to 37 degrees Celsius to the pellet and resuspend gently by pipetting with pipette tips or glass pasture pipettes prewarmed to the same temperature.
Incubate at 37 degrees Celsius for 10 minutes and then pellet the cells at 6, 000 G for one minute. Solidify the gelatin on ice for 30 minutes. To remove the gelatin embedded cells from the tube, cut the tube end containing the pellet off from the rest of the tube with a razor blade.
Then cut the tube end with the cell pellet in half perpendicular to the first cut. Place the two tube end halves containing the cell pellet in 2.3 molar sucrose and incubate for 10 minutes at four degrees Celsius. This causes the cell pellet halves to shrink slightly and dislodge from the plastic tube.
Remove the tube halves with the gelatin embedded cell pellet from the sucrose, and then remove the cell pellet halves from the plastic tube halves with tweezers. Manually cut the pellet into blocks of an appropriate size with a razor blade, and use a stereo dissecting microscope to magnify the subject during the cutting. Incubate the cell blocks overnight in 2.3 molar sucrose at four degrees Celsius.
Place them on an aluminum sample holder pin. Leave enough sucrose around the edges of the block so that it forms a thin collar between the block and the pin. Snap freeze and store it in liquid nitrogen.
Trim the front of the block to flatten its surface to obtain around 250 nanometer thick sections. Then trim the sides of the block by sectioning 50 to 100 micrometers into the side of the sample block face with the knife corner. Trimming four sides of the sample block face will create a protruding around 250 by 375 micrometer rectangle.
Section a ribbon from the protruding rectangle ensuring the sections are between 70 and 90 nanometers in thickness and have a silvery golden sheen. Guide the sections away from the diamond knife's edge with a hair on a stick to create a long ribbon. Pick up the ribbon by dipping the three millimeter pickup loop into a one-to-one mixture of 2.3 molar sucrose and 2%methylcellulose.
Insert the loop into the cryo chamber of the microtome, and once the droplet starts to freeze, promptly pick up the sections by gently pressing the droplet against them. Remove the loop from the cryo chamber. Wait until the droplet has thawed completely and press the droplet on a prepared grid.
Place the grids with sections on around one milliliter of PBS in a small dish or multi-well plate and incubate at 37 degrees Celsius for 30 minutes. Process the grids on around 75 microliters of droplets on Parafilm and start with PBS glycine washes at room temperature. Incubate the grids with a blocking buffer made of 0.1%bovine serum albumin C and 0.5%fish skin gelatin in PBS for 10 minutes at room temperature as the blocking step.
Dilute primary antibodies in blocking buffer in PBS and incubate the grids on around 10 microliter droplets of this solution for one hour at room temperature. Wash the grids in 0.1%bovine serum albumin in PBS five times at room temperature. Dilute the secondary antibodies and DAPI in the blocking buffer in PBS.
Then incubate the grids on around 10 microliter droplets of this solution for 30 minutes or longer at room temperature before washing the grids in PBS five times as shown earlier. To mount the samples for light microscopy, submerge the grids in 50%glycerol in distilled water twice for five minutes at room temperature. Place one grid per cover slip between a glass slide and a cover slip in 50%glycerol, ensuring that the sections are facing the cover slip.
For light microscopy, take a glass slide with the sandwiched grids to a wide field microscope with an automated stage. Select a high magnification oil objective and create an image tile set of the ribbon of sections. Next, for unmounting and electron microscopy or EM contrasting, add 10 microliters of distilled water to the side of the glass slide cover slip sandwich, and wait for capillary action to fill the glass cover slip sandwich interface.
Carefully remove the cover slip without mixing immersion oil into the glycerol. Retrieve the grids with tweezers and submerge them in distilled water three times at room temperature to wash off the 50%glycerol. Carefully dry the back of the grid with a lint-free tissue paper and place the grids section side down on distilled water droplets.
To stain the sections for contrast in electron microscopy, incubate them with uranyl acetate for five minutes at room temperature. Cool the uranyl acetate methylcellulose solution by placing the droplets on Parafilm on a metal plate on ice. Then wash the grids with this ice cold solution twice and incubate them in the solution for 10 minutes.
Loop out the grids by inserting a grid drying loop into the uranyl acetate methylcellulose droplet below each grid and gently lifting it until the grid is pulled off the droplet. To blot the excess solution, touch the loop at around a 60 degree angle onto the lint-free filter paper and drag it slowly along the paper until no more solution is absorbed. Place the loop with the grid in a suitable rack, and let it dry at room temperature for more than 10 minutes.
Use the overview obtained by light microscopy to locate a region of interest for imaging in the transmission electron microscope or TEM, and annotate the ROI on the light microscopy dataset. Once a region is selected, obtain an image tile set at 20, 000x to 50, 000x magnification in the TEM. Reconstruct the image tile set in post-processing software.
Perform the correlation based on DAPI signal and fluorescence and nuclear outlines in electron microscopy. Shift the images to overlay them precisely and perform the manual correlation accurately. Hepatoblastoma derived HEPG2 cells were starved for two hours in EBSS prior to fixation with 4%paraldehyde for two hours.
IF imaging of LC3 and LAMP1 on sections reveals relatively few LC3 puncta and little colocalization with LAMP1. Molecular information from IF was linked to the ultra structural information obtained in electron microscopy by overlaying the two imaging modalities based on DAPI and nuclear outlines. The correlation of the LC3 positive organelles to EM ultrastructure revealed that the different puncta represented distinct stages of autophagy.
The ultrastructure of the individual LC3 labeled compartments as exemplified by box one is shown here. The correlative light electron microscopy images are shown on the left, and the pseudo colored electron microscopy images are shown on the right. Inner and outer autophagosomal membranes are indicated by white and black arrowheads.
Interestingly, EM identified relatively weak fluorescent spots as LC3 positive autolysosomes, which are characterized by dense content and intraluminal vesicles. This reveals minimal LC3 visibility in IF of ultra thin cryo sections, suggesting detectable LC3 in steady state autolysosomes despite the degradative environment. However, LC3 positive puncta mainly represent autophagosomes with few representing autolysosomes.
For researchers who want to do immunogold labeling instead of on-section CLEM, the protocols and tips described here are fully applicable for immunogold labeling as well. We initially applied on-section CLEM to endosomal regulatory proteins of low abundance and showed for the first time, their endogenous ultrastructural localization.
