Protocol for fixation, embedding, sectioning, and staining of embryos

Below we describe our current protocol for the processing of maize kernels for paraffin sectioning of embryos. This protocol is a revised and more detailed version of that previously reported (J.K. Clark and W.E. Sheridan, J. Hered. 77:83, 1986).

Preparation of kernels and fixation: Immature and mature kernels are removed from freshly harvested ears and the sides of the kernels are trimmed with a razor blade in order to facilitate penetration of the fixative. The kernels are fixed in FAA [by volume, 60 parts 95% ETOH, 20 parts 20% formaldehyde, 5 parts glacial acetic acid, and 15 parts distilled water]. After 24 hours or longer of exposure to the fixative the vials containing the kernels are placed in a dessicator under vacuum, in order to remove any air remaining in the kernels.

Dehydration and embedding: Kernels are dehydrated by the tertiary butyl alcohol (TBA) procedure (D.A. Johansen, Plant Microtechnique, 1940). It is important to leave the kernels for an adequate time period for each step in the dehydration series; we use a 48-hour minimum time for each step until the kernels are moved into the oven, and then 8 to 24 hours for each step thereafter. The dehydration and embedding steps are as follows: one each of 20% ETOH and 30%, ETOH, followed by 2 48-hour exposures to each of the following:

Grade 1 50% ETOH, 10% TBA, 40% distilled H20
Grade 2 50% ETOH, 20% TBA, 30% distilled H20
Grade 3 50% ETOH, 35% TBA, 15% distilled H20
Grade 4 50% ETOH, 50% TBA, saturated with Eosin Y
Grade 5 25% ETOH, 75% TBA, saturated with Eosin Y
 
We then place the vials on top (outside surface) of the embedding oven and provide 2 48-hour exposures to 100% TBA saturated with Eosin Y, followed by a 48-hour exposure to a mixture of equal parts of TBA and paraffin oil. The kernels are then placed in a small volume of fresh TBA:paraffin oil (1:1) and poured into fresh vials half filled with warm semi-hard paraffin wax. These vials are placed in the 60C paraffin oven. After 8 hours (or overnight) the fluid is poured off and the kernels are exposed to 2 changes of paraffin wax of 8 to 12 hours each. This is followed by replacement of the wax with Paraplast plus for 8 hours, a change of Paraplast plus for 24 hours, and a final change of Paraplast plus for 48 hours. The kernels are then embedded in fresh Paraplast plus using Peelaway plastic models to cast the blocks. Casting is facilitated by use of a hot plate and slide warmer to keep the Paraplast plus molten and to allow arrangement of the kernels in the mold prior to placing on the surface of ice water (See Johansen for details). We orient the kernels so that the embryonic axis is facing downward toward the bottom of the mold.

Sectioning and Mounting: The blocks of Paraplast plus are trimmed so as to provide a 2-3mm margin of paraffin around the edges of the kernel, and then are attached with the aid of a heated dissecting needle to the end of a small oak block which has been previously soaked in melted paraffin. Sections are cut on a rotary microtome (see S.E. Sass, Stain Tech. 20:93, 1945), to obtain 15 micrometer thick serial sections. These ribbons are arranged on black construction paper in necktie boxes and scanned under a dissecting microscope. Sections lacking embryo material are discarded and the remaining sections are cut to convenient lengths and are mounted onto glass slides. A drop of Haupt's adhesive (A.W. Haupt, Stain Tech. 5:97, 1930) is spread on the surface of the slide using a fingertip and the surface is flooded with a 3% formalin solution, in order to float the ribbons. After gentle warming of the flooded slides so that wrinkles in the ribbons flatten out, they are drained and allowed to dry at least 24 hours.

Staining and coverslipping: The slides bearing the mounted sections are passed through 2 changes of xylene, one change of xylene:TBA (1:1) and 2 changes of 100% TBA, to remove the Paraplast plus and prepare the sections for staining in aqueous buffer. The slides are passed through 3 or more changes of distilled water (until the odor of TBA is no longer evident), and then one change of 0.025M citric acid-sodium phosphate buffer, pH 4.0. They are then placed in 0.005% Toluidine Blue O in the same buffer (pH 4.0) for 4 to 15 minutes. During staining the slides are periodically removed from the staining solution and examined under a dissecting microscope using transmitted light, to evaluate the staining intensity. When the sections are adequately stained they are rinsed for 1 minute in distilled water and air dried overnight. Overstained slides may be destained in a 1:1 mixture of TBA:Buffer followed by washing in the buffer before restaining. The air-dried slides are passed through 2 changes of xylene into clean xylene and coverslips are placed over 3 drops of Preserv-a-slide by gently lowering them from one end with a clean dissecting needle while holding the other end. The stained sections are photographed with Kodak Technical Pan 2451 which is developed with Technidol or Dektol.

We have had good success with this protocol in sectioning kernels aged from 0-80 days after pollination. For very young kernels we increase staining time. Older kernels frequently require a longer time in fixative, and an increase in the time allowed for the dehydration and embedding steps. Kernels may be stored in the FAA solution indefinitely. Note that TBA crystallizes at temperatures below 25C; therefore we add normal butyl alcohol (1 part to 99 parts TBA) to prevent this.

William F Sheridan, Janice K. Clark, Guy Farish and Shelley M. Horne


Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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