IHC Protocol for Free Floating Brain Sections

Identifying multiple proteins within the same tissue allows for assessing protein colocalization, is cost effective, and maximizes efficiency. Here, we describe a protocol for multiplex immunolabeling of proteins in free-floating brain sections. As opposed to slide-mounted immunohistochemistry, the free-floating approach results in less tissue loss and greater antibody penetration. Using distinct fluorophores for individual proteins, this protocol allows for visualization of three or more proteins within tissue sections. The protocol can be applied to other tissue types.

• Coronal 30-40 µm sections cut on a freezing microtome. Sections collected into petri dishes containing 1-2 ml 0.1 M phosphate buffer (PB). Sections can be kept on a shaker at 4°C for several days before commencing the immunocytochemistry.
• Deactivate endogenous peroxidases (For 40 ml, 20 ml 0.2 M PB, 8 ml methanol, 80 µl triton-X100, 2 ml hydrogen peroxide and make up to 40 ml with ddH₂O). Incubation for 15 min.
• Wash (3 x 15 min) in 0.1 M PB/0.3% triton.
• Preincubate in 0.1 M PB/0.3% triton/1% blocking serum for at least 1 h.
• Incubate at 4°C in primary antibody.
• Wash (3 x 15 min) in 0.1 M PB/0.3% triton.

• Secondary antibody either 2 h, room temperature (1:200) or overnight at 4°C @ 1:500 (made up in buffer 0.1 M PB/0.3% triton/1% blocking serum).
• Wash (3 x 15 min) in 0.1 M PB.
• Wash once in 0.1 M acetate buffer-briefly (just to remove PB).
• Place sections in solution for DAB reaction (see below). Monitor DAB reaction on microscope (2-10 min).
• Stop reaction once background is high enough by placing sections into 0.1 M acetate buffer. Finally, 3 washes in 0.1 M PB. Sections can be kept in cold room on share for a couple of days, or until mounted and cover-slipped.

• If left too long, the sections become harder to mount.
• Acetate buffer must be made up fresh.
• The 0.1 M PB should be no triton.

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