Recommended strategy for anti-HA staining

We've characterized a number of antibodies and piloted many protocols for visualizing HA-tagged DREADDs.

Here's our recommended protocol (please note suppliers of antibodies for best results):

Immunofluorescence Staining Protocol (Anti-HA antibody)
1. Perfuse the mice with ice cold 4% paraformaldehyde (PFA; freshly prepared)

Cryosection procedure:
2. Fix the brain with 4%PFA @ 4°C overnight (O/N).
3. Transfer brain sample to 30% sucrose/1x phosphate buffered saline (PBS), allow sample sink @ 4°C for about 48hr.
4. Cryosection 40 uM

Staining procedure:
5. Permeabilize the tissue with 1xPBS/0.4% Triton x100 for 1hr at room temperature (RT).
6. Block the slide with 1% BSA 5% NGS (normal goat serum) in 1xPBS/0.4% Triton x100 at RT for 1hr.
7. Incubate the sections with primary antibody (1:500) in blocking buffer @ 4 O/N.
8. Wash the section with 1x PBS/0.4% Triton x100 three times at RT, 10min/each.
9. Incubate the sections with secondary antibody (1:250) in blocking buffer for 2hr at RT.
10. Wash the sections with 1x PBS/0.4% Triton x100 three times at RT, 10min/each.
11. Mountain the section with aqueous mounting medium.

Primary antibody: Cell signaling, HA-Tag (C29F4) Rabbit mAb, Cat # 3724

Secondary antibody: Invitrogen, Alexa Fluor® 488 goat anti-rabbit IgG (H+L), Cat# A-11008

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