Expansion microscopy

This protocol was used to expand the samples in a way that the finer arborizations, that usually cannot be resolved by standard sample preparation and imaging techniques could be imaged and quantified.

Physical expansion of the sample was achieved by several steps; perfusion of the sample by an

acrylic-37 based gel, gelation (or gelling) of the sample, digestion of the proteins in the brains that keeps cells intact, and finally the expansion of the gelated sample. I have utilized the protocol from Asano et al.

(2018) with small modifications.

Preparation of gelating chamber;

In the study of Asano et al. (2018), gelation chambers were prepared during gelation. However, since they were not stable, in this study, a slightly different version of gelation chambers were prepared before the expansion microscopy experiments. Gelation chambers were prepared such that six pieces of sellotape were stretched on top of each other horizontally over a thick glass slide (Figure 2.4A) and 3 equally distributed slits (app. 2-3mm wide) were made with the help of a razor blade orthogonal to the tape layers (Figure 2.4B). Six layers of sellotape measured at approximately 500-600µm in height, perfectly fitting the fly brain, at approximately 500µm in depth, were used. Each slit contained a single brain and was covered by single plastic coverslip. Plastic covers were preferred as they were less sticky after the gel was polymerized and were easy to remove. Additionally, gelation chambers used in this study could be washed for continued use in further experiments.

Figure 2.4 Illustration of the gelling chamber and gelation of the brain. A Top view of gelation chamber. Six pieces of sellotape were positioned on top of each other and three equally distributed slits are opened with a razor. B Side view of gelation chamber. Height of the slits are slightly higher than the fly brain. C Positioning of the fly brain inside the gelling solution. The anterior part of the brain was placed in a downward position, slightly touching the glass surface of the slide. D Illustration of the carved gel block containing a gelated brain. The gel block was trimmed from the corner to orient the anterior side.

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Dissection and IHC;

Dissection and fixation of the female fly brain was performed exactly the same as it is described in 2.2.8.

However, unlike the “regular” IHC protocol, the primary antibody was always used at twice the usual concentration, i.e. anti GFP (chicken) was diluted 1:500 in blocking solution instead of 1:1000, and the brains were incubated overnight at 4°C followed by room temperature incubation for 5 h on the shaker.

The next day, the primary antibody was washed in the same way as the fixative and the solution was replaced by 1:200 diluted secondary antibody (instead of 1:300) overnight at 4°C in darkness on a shaker. From this step on, the samples were kept in darkness as much as possible. Finally, the secondary antibody was washed using PBS (instead of PBS-T) for the further expansion microscopy protocol.

Gelation of the sample;

In order to prepare the brains for perfusion with gelling solution, brains were incubated in AcX/DMSO solution in MBS for 24 h at room temperature in darkness (without shaking) in black glass dishes. Then, AcX/DMSO solution was washed with PBS three times for at least 20 min. Then, the samples were chilled on ice for a minute before being transferred into the gelling solution. The gelling solution was prepared the same way as described in Asano et al., 2018. Components of the gelling solution and the ratio of their volume in the solution are following; StocX, 4HT, TEMED, and APS with the ratio of 47:1.5:1:1 respectively. The gelling solution had to be freshly prepared and had to be used immediately after the last component is placed in the solution. The order of the components must be the same as stated above and the solution vortexed thoroughly after the last component is placed. Subsequent to washing ofo AcX/DMSO, the gelling solution was applied and the samples chilled further on ice for 60 minutes in darkness without shaking. Then, the brains were transferred into the gelating chamber that is described above. Prior to the transfer, approximately 20-50µl of gelation solution was pipetted into each slit in a way that the height of the solution was not above the tape walls. Then, the brains were transferred into the gelation chamber and positioned in a way that the anterior position was facing downwards and touching the bottom of the slit. Transferring and mounting of the brain into the gelation chamber had to be as quick as possible because since addition of the last component of gelling solution, APS, initiated the polymerization at room temperature. Finally, each sample was covered by plastic coverslips, making sure that each brain was covered entirely by gelling solution and that no air bubbles were created after the placing of the coverslips. The gelation chamber was placed in a 37°C incubator for 2 hours in darkness for polymerization. Subsequent to polymerization, coverslips were carefully peeled away and a small block of gel should be cut with the help of a razor blade (Figure 2.4C).

At this stage the brains were visible in the gel and during cutting, a visible amount of gel surrounded

39 the gel. The gel block could also be trimmed in a way that the orientation of the brain was predictable (Figure 2.4C).

Digestion;

The digestion solution was also prepared freshly since it contains digestive enzymes. During gelation, digestive 800U proteinase K containing digestion buffer was prepared on ice. After cutting the gelated brain blocks, each block was transferred into 200µl of digestion solution in single wells of a 96 well-plate using a paint brush. Prior to transfer, a drop of digestion solution was put on the block to make transfer easier. The 96 well-plate was covered with aluminum foil to limit light exposure. After transferring each brain, the lid was closed and the plate was sealed tightly with parafilm to prevent evaporation. Samples were left at room temperature without shaking for 3d for digestion.

Expansion;

During the digestion period, the samples already started expanding by a factor of 1.5 to 2. Following 3 days of incubation for digestions, the digestive solution was replaced by water and samples were incubated in water for 40 min, refreshing the water halfway through.

Mounting for imaging;

All the samples were mounted and imaged immediately following expansion. Each gel block was placed on a glass slide and excessive amount of water was dried with tissue paper. Then, three component dental glue was mixed and used to surround the gel blocks using a toothpick. Since at this stage the brain was invisible, brains could be oriented such that the antennal lobe, hence also the MB lobes, were in an upward position by the help of trimmed shape of the gel block. A drop of water was placed on top of the gel block to prevent it from drying.

Imaging and colocalization analysis;

Following the stabilization of the gel blocks, the samples were imaged using a 10X air objective since the working distance allowed visualization of the expanded samples at such depth. The imaged sample files and imaging settings were saved as “.lif” and “.seg” files, respectively. Images of the samples were analyzed with Image J software. The images were smoothened using the mean filtering function with the factor 1 to refrain from a rough picture since the samples were expanded much above the optical spatial resolution. A threshold was applied to the images to eliminate background fluorescence.

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Colocalization of the proteins of interest for quantification was then analyzed using the image calculator function. To do this, the two image channels of the same brain (each corresponding to a different proteins of interest) were separated as two separate images. The AND function of image calculator was then used between the two images in order to detect the co-localized pixels that showed a fluorescent signal in both channels.