Tissue Fixation Embedding Sectioning And Mounting Of Sections On Slides


1. Label 10- or 20-ml snap-cap glass vials and their caps according to the samples, and fill 2/3 volume of each vial approximately with freshly prepared 10% buffered-formalin or 4% PFA fixation solution.

2. Place freshly dissected tissues, organs or embryos in the appropriate vials and allow fixation to proceed at 4°C for an optimal time period.

Note: Tissue fixation is a key step for the success of in situ hybridization and the desired fixation time varies with tissues. For good morphology and a good signal-to-noise ratio following in situ hybridization, an optimal time should be determined empirically. Based on our experience, series of fixation times ranging from 30 min to 15 h should be set up for each sample.

3. While the samples are being fixed, melt an appropriate amount of paraffin wax in a clean glass beaker in a 60°C oven.

4. After fixation, carry out dehydration of samples by carefully removing the fixation solution from the vials and replacing it with 10 ml of 50% ethanol.

5. Perform complete dehydration by transferring the slides every 20 min through an ethanol series (50%, 70%, 95%, 100%, 100%, and 100%) and 2 x 15 min in xylene.

Note: Tissue dehydration must be as complete as possible. Otherwise, melted paraffin wax may not thoroughly penetrate the tissue, resulting in poor paraffin blocks.

Caution: Xylene is a toxic solvent and should be carefully handled. It is strongly recommended that any steps with xylene be carried out in a hood. Used xylene should be collected in a special container for toxic waste disposal.


1. Following the previous step 5, carry out partial impregnation of the samples by quickly filling each vial with 4 ml of freshly mixed xylene and melted wax (1:1, v/v) using a hot glass Pasteur pipette. Allow incubation to proceed overnight at room temperature.

2. Melt the mixture in a 60°C oven and immediately replace the xylene/wax mixture with fresh melted wax. Allow impregnation of the samples to proceed at 60°C for 1 h.

3. Continue wax infiltration of the tissue samples by quickly replacing with fresh melted wax every 20 min using a hot glass Pasteur pipette. Repeat this step 10 times. The samples are ready for embedding.

4. Prepare embedding boats using aluminum foil or molds according to the manufacturer's instructions. Appropriate molds and boats are also commercially available.

5. Fill embedding molds with melted paraffin wax using a hot glass Pasteur pipette and quickly transfer samples to appropriate molds using a hot cutoff Pasteur pipette. Orient the samples within the mold with a hot drawn-out and sealed Pasteur pipette.

Note: This step should be performed gently to avoid any bubbles that may become trapped inside the blocks. Place one sample in each block. Every embedding boat or mold should be clearly labeled for sample identification.

6. Allow cast blocks to harden completely at room temperature or place boats or molds into cold water in a tray.

7. Remove the blocks from the embedding boats or molds and store the blocks in a dry place at room temperature until sectioning.

Note: It is important to store the blocks in a dry place. Otherwise, moisture may ruin the paraffin wax blocks. The blocks can be stored for years under these conditions.

Sectioning and Mounting

1. Using a razor blade, carefully cut each of the paraffin wax blocks containing the embedded samples into a trapezoidal shape and continue to trim the sectioning surface until approximate 1 to 2 mm from the sample.

2. Place the trapezoid block in the holding clamp of a microtome with the wide edge of the trapezoid surface facing the knife.

3. Set up the thickness of sectioning at 4 to 10 mm according to the manufacturer's instructions for a particular microtome and start to cut section ribbons.

4. Label previously coated glass slides according to sample blocks and add one to three drops of dd.H2O or 0.2% (w/v) gelatin subbing solution to a slide. Place the slide onto a heating plate set at 50°C.

5. Carefully transfer the ribbon of sections (3 to 10 sections) onto the gelatin-or water-drop on the slide using a fine brush. Allow the ribbon of sections to stretch to remove any wrinkles until complete drying of the slide has occurred (Figure 12.1).

6. To attach the sections to slides firmly, place the slides at 40°C for 24 h.

7. Store the slides in a slide box with desiccant at -20°C until use.

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