- I have never worked with brain very much and would love some insight on how to prepare it before IF and a protocol to start from. Thank you much.
Here is a general protocol that you can use as a starting point:
Mice are anesthetized and perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains are removed and post-fixed in the same fixative for 2-4 hours at 4°C.
Brains are cryoprotected by incubating in 30% sucrose in PBS until they sink. Brains can be frozen on dry ice or in a cryostat and cut into 20-30 µm thick coronal sections.
Sections are washed in PBS, permeabilized with 0.3% Triton X-100 in PBS for 30 minutes, and blocked with 5% normal serum (e.g., goat or donkey serum) in PBS for 1 hour at room temperature.
Primary antibodies against specific antigens of interest (e.g., PER1, BMAL1, or CLOCK) are applied to sections and incubated overnight at 4°C.
Sections are washed in PBS and then incubated with fluorescently-labeled secondary antibodies for 1-2 hours at room temperature.
Sections are washed in PBS, mounted onto slides with a mounting medium containing a nuclear counterstain (e.g., DAPI), and cover-slipped.
Slides are imaged with a fluorescence microscope or confocal microscope.
This protocol can be modified based on the specific antibodies and detection systems used.
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