Rat anti mcherry

Perfusion and histology were performed as described in Garfield et al.^{153 (link)}. Brain slices (60 μm thick) were collected and one of every three consecutive slices was scanned. The primary antibodies used in this paper are chicken anti-GFP (1:1000, Invitrogen; used for GFP and GCaMP6s) and rat anti-mCherry (1:1000, ThermoFisher; used for mCherry and tdTomato). The secondary antibodies are Donkey anti-chicken 488 (1:1000, Jackson) and Donkey anti-rat 594 (1:1000, Jackson).

Brain slices were fixed in Roti-Histofix (PO873, Carl Roth) for ~12 h at 4°C and subsequently rinsed in 0.1 M phosphate-buffered saline (PBS, 3 × 10 min). PBS contained (in mM) 72 Na₂HPO₄ x dihydrate, 28 NaH₂PO₄ monohydrate, resulting in pH 7.2. To facilitate antibody penetration and prevent unspecific antibody binding, brain slices were preincubated in PBS containing 1% (w/v) Triton X-100 (TX, A1388, AppliChem) and 10% (v/v) normal goat serum (NGS, ENG9010–10, Biozol Diagnostica) for 30 min at room temperature (RT). Brain slices were then incubated for ~20 h at RT with primary antibodies (chicken anti-GFP, 1:1000, ab13970, Abcam; rat anti-mcherry, 1:1000, Thermo Fisher Scientific, M11217) in PBS-based blocking solution containing 0.1% TX, 10% NGS and 0.001% sodium azide (S2002, Sigma-Aldrich). Brain slices were rinsed first in PBS-0.1% TX (2 × 10 min, RT), then in PBS (3 × 10 min, RT) and subsequently incubated with secondary antibodies (goat anti-chicken-FITC, Jackson #103–095-155, 1:500; goat anti-rabbit Alexa-Fluor-594, Thermo Fisher Scientific, A11012; 1:500) and DAPI (1:1000) for 2 hours at toom temperature. Brain slices were then rinsed in PBS-0.1% TX (2 × 10 min, RT) and PBS (3 × 10 min, RT), dehydrated in an ascending ethanol series, cleared with xylene (131769.1611, AppliChem), and mounted for imaging.