This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).
The overall goal of the hybrid-cut tissue sectioning method is to improve the integrity of sectioned recalcitrant plant tissues, those with tough fibers and silica crystals, to create suitable tissue samples for anatomical and biological studies. Plants that contain high level of crystal and silica can suffer severe paraffin tear and problem during sectioning. This method significantly improve tissue integrity.
The main advantage of this technique is that the sectioning under ultra low temperature in the cryostat hardens the paraffin block, significantly reducing the crystal shearing problem. The application of this technique can extend to in situ hybridization, FISH, and internal assess among others. Visual demonstration of this method is critical as the steps to ensure the paraffin block is adhered tightly to a stage can prove challenging.
To begin, select mature phalaenopsis orchid plants at the five leaf stage. Carefully remove the leaves by tearing them along the mid rib. Using a sharp scalpel, carefully remove the axillary buds from the base of the third or fourth leaf on the monopodial system.
Immediately place the samples into a glass scintillation vial containing 15 milliliters of ice cold PFA fixative. Apply a vacuum to the samples in the fixative at four degrees Celsius for 15 to 20 minutes. Small bubbles will be released from the samples.
Repeat this step until most of the samples sink after the release of the vacuum. Finally, hold the vacuum overnight and release it slowly the following day. Next, immerse the fixed samples in 15 milliliters of PBS and place on ice for 15 minutes.
Dehydrate the samples in 15 milliliters of ethanol in a series of increasing concentrations. 30%50%70%85%95%and finally 100%Infiltrate the samples with 15 milliliters of a two to one volume to volume ethanol and xylene substitute mixture at room temperature. Continue the infiltration by sequentially placing the samples in 15 milliliters of each of the following.
A one to one volume to volume mixture of ethanol and xylene substitute, a one to two volume to volume mixture of ethanol and xylene substitute, and finally, pure xylene substitute. Then incubate the tissues in an oven at 60 degrees Celsius. Infiltrate the samples with 15 milliliters of xylene substitute and paraffin mixture at 60 degrees Celsius.
Finally, infiltrate paraffin in 15 millimeters of pure paraffin twice for 12 hours each at 60 degrees Celsius. Repeat this step the following day. To begin embedding, first warm up the metal molds to 62 degrees Celsius, and then pour approximately 13 milliliters of molten wax into the mold base.
With warmed forceps, transfer a tissue sample into the mold and orient it into the desired position. Move the mold carefully onto the cooling plate and leave it to stand until the wax is solidified. Next, use a razor blade to trim the paraffin sample block into an appropriately sized column with a trapezoid surface at the top.
Add 800 microliters of optical cutting temperature compound, or OCT, to the center of the cryostat stage. Place the paraffin block onto the stage, and quickly orient the tissue block to the desired position. Transfer the paraffin block and stage to a crystat chamber.
Quick freeze the OCT at negative 42 degrees Celsius for 10 minutes. Next, cool the crystat adapter and chamber temperature to negative 20 degrees Celsius, and negative 16 degrees Celsius respectively. Attach the block and stage to the crystat adapter.
Now, section the tissues to 10 micrometers in thickness. Using forceps, pick up the tissue sections and place them so that they float on 800 microliters of DEPC treated water on a poly-l-lysine coated slide. Transfer the slides onto a hot plate set to 42 degrees Celsius.
Allow the sections to flatten and use filter paper to soak up any water from the edges. Leave the sample on the hot plate at 42 degrees Celsius overnight to allow the tissue to mount to the slide. To deparaffinize the samples, first place the mounted tissue slides into a staining rack.
Add 150 milliliters of xylene into a staining jar, and immerse the rack of slides for five minutes. Next, rehydrate the samples by placing them in decreasing ethanol concentrations, 150 milliliters each for three minutes at a time at room temperature. Finally, immerse the specimens in double distilled water for three minutes.
Stain the samples by placing the slides in 150 milliliters of hematoxylin solution for 1.5 minutes. Rinse for a few seconds in 150 milliliters of double distilled water containing one to two drops of N hydrochloric acid, and then wash the slides briefly with double distilled water. Dehydrate the samples once more using a series of 150 milliliters of increasing ethanol concentrations for three minutes at room temperature.
Immerse the specimens in 150 milliliters of xylene for five minutes. Finally, mount the samples by gently adding 600 microliters of xylene-based mounting medium onto each slide, and then placing a cover slip over the specimen. Using this method, tissue sections of phalaenopsis orchids were generated, including the typically difficult to section axillary bud, which contains high levels of crystals.
Detailed structures were preserved, allowing visualization of protein bodies. The hybrid cut also produced clear sectioning for visualization of the shoot apical meristem of the germinated seed. These images show the preparation of transverse leaf sections of rice and wheat plants using the traditional paraffin versus hybrid cut sectioning techniques.
These crops typically contain high levels of silica, which makes them challenging to section. The tissue integrity in the hybrid cut sections was significantly improved, and key structures often distorted, or hard to find in paraffin samples, were preserved in their entirety in the hybrid cut tissue. Additionally, tearing was only observed in the paraffin sectioned samples.
Additionally, the strong retention of anatomy makes hybrid cut tissues useful for studies of spatial gene expression patterns. Here, in situ hybridization with Actin and Cyclin B1:1 was carried out on hybrid cut sections of the second and third axillary buds of the phalaenopsis orchids. The improved anatomical preservation allowed detection of higher expression levels of the target genes in the developing meristematic cells of the third axillary buds.
While attempting this procedure, it's important to maintain a temperature at minus 16 degrees centigrade to harden the paraffin block. This should result in good quality tissue section. We first had the idea for this method after we failed many times to perform successful sectioning in orchid using the traditional paraffin or cryosection method.
Following this procedure, all experiments like in situ hybridization and FISH can be performed in order to answer additional questions, including investigation of gene spatial localization and function. After watching this video, you should have a good understanding of how to perform high quality tissue sectioning in recalcitrant plant sample.
