This article details a protocol for preparing rat brain hemislices to study neurotoxic effects of peptides. The methodology includes tissue preparation, incubation, and analysis of protein accumulation related to neurodegeneration.
Take a rat coronal brain section and cut it along the midline to obtain hemislices, ensuring both originate from the same anatomical region for control and test experiments.
Transfer the hemislices to oxygenated aCSF for recovery.
Next, place them in vials with aCSF for the control and aCSF with a neurotoxic peptide for the test.
Seal the vials with oxygenating needles to supply oxygen to the tissue and incubate.
In the test vial, the neurotoxic peptide binds to its neuronal receptor, disrupting normal neuronal signaling.
This disrupted signaling leads to neurotoxic protein accumulation and alters microtubule-associated proteins, promoting neurodegeneration.
After incubation, transfer the hemislices to tubes containing chilled lysis buffer.
Homogenize the tissue to release cellular contents, while inhibitors in the buffer prevent protein degradation.
Centrifuge and collect the supernatant containing soluble proteins and store them for further analysis.
Carefully divide each section with small scissors along the midline to obtain two specular hemi-slices. Use each pair of hemi-slices for the control and treated condition. After being divided, let the hemi-sections rest in the vibratome chamber for 5 to 10 minutes.
After the rest, gently transfer the hemi-sections using a glass pipette into a bubbling pot containing recording aCSF at room temperature. Then, let the hemi-sections recover for about 30 minutes before proceeding. During the break, load three glass vials with 3 milliliters of recording aCSF for the control group and three vials with 3 milliliters of aCSF enriched with T30 for the treated group.
After the 30-minute rest, within 5 minutes, transfer the sample to the baths. First, load the consecutive hemi-slices to the control solution using a brush. Then, using a different brush, transfer the contralateral hemi-sections to the treatment solution.
Next, seal the vials with plastic stoppers containing 21-gauge needles for the delivery of carbogen to the sample. Deliver oxygen at a minimal flow rate, such as 2 milliliters per minute, to prevent any movement of the brain tissue, resulting in possible mechanical damage to the tissue. This completes the setup. Now, start a 5-hour incubation.
After the incubation, stop the oxygen flow and uncap the vials. Then, gently transfer the hemi-slices using the appropriate brush to 1.5-milliliter tubes containing 250 microliters of lysis buffer, maintained on ice. Next, using separate pestles, homogenize each tissue. Complete the transfer and homogenization steps within 10 to 15 minutes for all six samples.
Now, centrifuge the homogenized samples at 1,000 times gravity for 5 minutes at 4 degrees Celsius. After the spin, transfer the supernatants into new tubes and store the samples at negative 80 degrees Celsius.
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