- Remove fixative solution and replace with 1 mL 30% sucrose.
Allow tissues to equilibrate for 2 h to overnight at 4C(see
Note 6). - Transfer the equilibrated tissue into a plastic embedding mold.
Orient the tissue with the ventricle flat on the bottom of the
mold. - With a Kimwipes, absorb as much of the sucrose solution as
possible. - Slowly add Tissue Freezing Medium to cover the entire tissue,
filling the mold to about¾of the way full. If necessary, reposi-
tion the tissue with Dumont #5 forceps. - Freeze the embedded tissue at 80 C for at least 2 h prior to
proceeding. Tissues may be stored frozen in blocks indefinitely
(seeNote 7).
3.2 Tissue
Sectioning
- Before sectioning, equilibrate the tissue block to the tempera-
ture of the cryostat chamber, at 25 C, for 30 min. - Section tissues at a thickness of 10μm on cryostat and transfer
sections onto Superfrost/Plus microscope slides (seeNote 8). - Allow sections to air-dry overnight at room temperature. We
generally add Drierite desiccant into the slide box with the
slides to facilitate faster drying. Slides can be used immediately
or stored at 20 C for up to 1 year. - When ready to perform the probe hybridization, warm the
slides at room temperature for 1 h. This is usually sufficient
time to ensure the slides are dry and warm.
3.3 Antigen Retrieval 1. Before proceeding to pretreatment, fill a Coplin jar with anti-
gen retrieval buffer and place the Coplin jar inside a 500 mL
beaker. Add water to the beaker until the water level reaches
~3/4 of the Coplin jar. Using aluminum foil, cover the entire
assembly. Take care to ensure that water is not splashing into
the antigen retrieval buffer.
- Place a thermometer inside the Coplin jar and apply heat in
increments until the citrate solution reaches a temperature of at
least 100C. If the solution is heated too quickly, the Coplin jar
will crack. While the antigen buffer is heating, proceed with the
following steps (seeNote 9). - Outline the tissue sections on each slide with a hydrophobic
pen in order to create a barrier to hold incubation solutions. Be
cautious to not touch the tissues. Allow the ink barrier to dry
completely. - Rinse the slides in 1PBS for 5 min in a Coplin jar. Gently
move slides up and down to remove Tissue Freezing Medium.
202 Viravuth P. Yin