Lab Schedule (3-hour lab)

1. (20min) Review steps performed by the instructor: perfusion, cyoprotection, and embedding in O.C.T.
2. (Groups will take turns observing, 15min each, ~ 1 hour total) Observing cryosectioning
3. (While groups wait) Review your Official Experiment protocol & preparation (Plan out of class dates/times)

TO PREPARE:

• Read and review this lab (there will be many techniques coming up today!)

DUE AFTER LAB:

• Show any of the instruction team your answers to the lab questions. This is comprehension check.

• Talk to the instructor about who is able to come in on a Monday for Lab 6.5

• Submit your Final IHC Protocol via Moodle

  🈴 Graded Component (25 points) - Rubric provided

1. Perfusion, Cryoprotection, & Embedding in O.C.T.

(These steps were done by the instructor outside of lab time)

1. The goal of perfusions is to clear the mouse brain of all the blood and then fix it to preserve it for later steps. To do this, we use the mouse’s own circulatory system to first pump saline to clear the clear and then to pump a 4% paraformaldehyde solution through. You can read the protocol here.
2. We then extract the brain using the same procedure you followed in Lab 4, expect that the brain is much less squishy and easier to work with.

1. After the brain tissue is extract, they sit in paraformaldehyde overnight in the 4*C fridge.
2. The day after we “cryoprotect” the brains using the following procedure:

Place tissues in 15% sucrose in PBS until tissue sinks (6-12hrs) and then 30% sucrose in PBS for overnight or until tissue sinks.