Spatially Compact Arrangement of Larval Zebrafish Sections for Spatial Transcriptomic Analysis

In the Morhardt Lab, we use zebrafish as an animal model to study bladder development and function. We've identified a functional urinary bladder that fills and empties in zebrafish. While zebrafish are small, we sought to take advantage of its size to economically utilize spatial transcriptomic techniques and our understanding of the vertebrate bladder.

Spatial transcriptomic techniques can quickly become expensive if reagents and equipment are not optimized. This protocol provides a proven technique to achieve this using zebrafish and provides the insights into the expected results and mitigation of problems that may arise as a result of the technology. Besides the economic utilization of expensive spatial transcriptomic technologies, this work has allowed us to perform large scale spatial transcriptomic analyses of essentially whole organs across several ages while minimizing batch effects.

To begin, cool the cryostat to 22 degrees Celsius and gather all the required items. Prepare a disposable plastic mold for sample alignment. Place a piece of lab tape on the inside of the base mold and draw a straight line across it with a permanent marker as a reference point.

Mark the inside of the base mold to ensure proper sample orientation during cryosectioning. After priming the nozzle of the freezing medium bottle, dispense the necessary amount into one corner of the base mold. Slowly tilt the base mold to evenly distribute the medium.

Now, place the base mold with freezing medium in an ice bath and incubate it for at least 10 minutes to cool the medium. Next, add one part of 100%ethanol to one part of dry ice in an ice bucket under the fume hood. Place the disposable aluminum dish or boat in the ice bath and cover the bucket for five to 10 minutes.

Collect the euthanized fish after submerging them in four degrees Celsius water for 10 minutes. Using fine-tipped forceps, grasp the caudal fin to remove the fish and gently press it against an absorbent lint-free wipe to dry it. Place the prepared base mold under a stereo microscope with 10x magnification.

Position the sample in the mold along the reference point in the correct orientation. Gently cover the sample with another thin layer of freezing medium. Use an anatomical reference point to align the fish precisely to the marked lines inside the base mold and adjust the orientation using fine-tipped forceps, avoiding bubbles.

Apply a piece of dry ice to the bottom of the base mold under the sample until it is locally frozen into position. Maintain a level horizontal plane to prevent sample misalignment before freezing completely. Now place the base mold with the sample onto the aluminum boat or dish in the dry ice and ethanol bath.

Ensure the boat remains floating at the surface and the base mold stays dry. Cover the bath and allow samples to freeze for 10 minutes. Next, wrap the frozen base mold in foil and store it in a freezer at 80 degrees Celsius until ready for sectioning.

Bring frozen base mold to the pre-cooled cryostat for cryosectioning and place it in the cryostat chamber. After removing the spatial imaging slide from storage, place it in a pre-chilled slide holder. Store the slide holder with the spatial imaging slide in the 22 degrees Celsius cryostat chamber until ready for section collection.

Now, remove the frozen sample from the base mold and place it in a chuck with fresh freezing medium, ensuring that the cutting surface faces the microtome blade. Then place a fresh fine microtome blade in the cryostat. After aligning the mold to the blade, trim the region of interest with a recommended thickness of 20 to 50 micrometers.

Ensure that markings inside the mold are transferred to the frozen block for proper sample orientation. Adjust the mold during trimming so that the cutting surface remains parallel to the reference markings in the freezing medium. Slice sections from the frozen block and collect them onto a standard positively charged microscope slide.

Check the sections under a brightfield microscope to confirm that trimming is complete. Then remove the spatial imaging slide from the cryostat chamber and place it in a four degree Celsius ice bath, while keeping it inside the slide holder. Begin cryosectioning at a recommended thickness of 10 to 14 micrometers.

Collect sections onto positively charged slides until the region of interest is reached. Now retrieve the single-cell spatial imaging slide from the ice bath and remove the slide from the holder. Collect sections row by row using a fine-tipped paintbrush to prevent rolling.

Then press a corner of the empty freezing medium onto the knife stage using the backside of a paintbrush. Use the top of the slide as a pivot point and slowly lower the slide onto the section to let it adhere for three seconds before lifting. Line serial sections parallel to each other to maximize space on the slide.

After collecting sections from the region of interest, place the slide back into the slide holder. If no more samples are needed, store the slide at 80 degrees Celsius for up to two weeks before analysis. Collect sections before and after the region of interest onto a standard positively charged microscope slide and perform hematoxylin and eosin staining to assess sample alignment and section quality before proceeding with the analysis.

Multiple section alignment was verified by comparing structures across sections in the same cut. Adjustments to embedding and collection steps enabled a greater number of sections to fit within the imaging area of a spatial transcriptomic slide. Spatial transcriptomic signal quality was high within tissue regions but lower in empty slide areas due to low signal complexity.