Immunohistology
Mai-Anh Vu, Mark Howe
Abstract
We have developed a new micro-fiber array approach capable of chronically measuring and optogenetically manipulating local dynamics across over 100 targeted locations simultaneously in head-fixed and freely moving mice, enabling investigation of cell-type and neurotransmitter-specific signals over arbitrary 3-D volumes . This protocol includes the immunohistology steps we used to confirm viral expression, and/or stain for neurons after our micro-CT scanning and contrast agent soaking procedure.
Steps
Removing Lugol's contrast agent from tissue
In preparation for immunohistology, Lugol’s solution was removed from brain tissue after CT imaging by soaking the implanted brains in a solution of 5% sodium thiosulfate (STS, Carolina) in 1% PBS for 4-6 days (Hopkins et al., 2015).
The implant was then removed, the brain returned to the STS solution for one hour.
Slicing
The brains were then moved to a solution of 30-40% sucrose in PBS.
Once the brains sank, coronal sections (50 µm) were sliced with a cryostat (Leica CM3050 S) and transferred to PBS for storage.
Immunostaining
To stain for dLight1.3b or ChR2 expression:
Sections were then blocked and treated in a 5% normal goat serum (NGS) and 0.2% PBS triton (Sigma Aldrich) solution (PBST).
Treated sections were then incubated for 24-48 hours at 4°C in 5% NGS, 0.2% triton, and a GFP primary antibody (chicken polyclonal antibody; 1:1000, ThermoFisher Scientific, No. A10262).
Slices were then washed, and incubated for 2 hours at room temperature in 5% NGS, 0.2% PBST, and an Alexa 488 secondary antibody (goat anti-chicken; 1:200, ThermoFisher Scientific, No.A-11039).
For neuronal staining:
Sections were then blocked and treated in a 5% bovine serum albumin (BSA) and 0.2% PBS triton (Sigma Aldrich) solution (PBST).
Treated sections were then incubated for 24-48 hours at 4°C in 5% BSA, 0.2% triton, and a NeuN primary antibody (guinea pig antibody; 1:500, Synaptic Systems, 266 004).
Slices were then washed, and incubated for 2 hours at room temperature in 5% BSA, 0.2% PBST, and an Alexa 647 secondary antibody (goat anti-guinea pig; 1:200, ThermoFisher Scientific, No. A-21450).
Mounting and imaging
Finally, stained sections were mounted onto glass slides using Vectashield Mounting Medium with DAPI (Vector Laboratories, H-2000).
Confocal images were acquired with a Zeiss LSM 800.
