Ab13970

PRV-injected sliced brain sections were removed from cryoprotectant and rinsed in PBS three times thoroughly at room temperature (RT) on a rocker for free-floating double-labeling fluorescent IHC. Non-specific staining was blocked with PBS containing 10% normal goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China, #AR1009), 2% Albumin bovine (BSA, BioFroxx, Germany, #4240G250G), and 0.4% Triton X-100 for 1h at RT on a rocker. The sections were then incubated with chicken anti-GFP (Abcam, USA, #ab13970; 1:1000) and rabbit anti-dsRed (Takara, USA, #632496; 1:1000) overnight at 4. The next day, sections were washed three times with PBS for 15 min and fluorophore-coupled with goat anti-chicken IgG Alexa 594 (Jackson ImmunoResearch, USA, #103-585-155; 1:500) and goat anti-rabbit (Jackson ImmunoResearch, USA, #111-545-003; 1:500) secondary antibodies for 2 h at 37. After 2 h, sections were washed twice with PBS containing 0.2% Triton X-100 (PBST) for 10 min and once with PBS. Finally, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime Biotech, Jiangsu, China, #C1002; 1:4000) was added to the stain cell nucleus for 10 min at RT, washed an additional three times with PBS, mounted with 70% glycerol on gelatin-coated glass slides (Citotest Scientific Co., Ltd, China; #80312316161), and sealed with nail varnish.

Mice were anesthetized with Beuthansia (0.2 ml, i.p.; Merck) and perfused transcardially with PBS followed by 4% PFA in PBS. Brains were post-fixed overnight in 4% PFA at 4 °C, cryoprotected in 30% sucrose, frozen in OCT compound (ThermoFisher), and stored at −80 °C. Coronal sections (30 μm) were cut on a cryostat (Leica Microsystems) and collected in cold PBS. For immunohistochemistry experiments, sections were washed three times in PBS with 0.2% Triton X-100 (PBST) for 5 min and incubated in blocking solution (3% normal donkey serum in PBST) for 1 h at room temperature. Sections were incubated overnight at 4 °C in blocking solution with primary antibodies including chicken-anti-GFP (1:10000, Abcam, ab13970) and rabbit-anti-dsRed (1:2000, Takara Bio, 632496). After 3 washes in PBS, sections were incubated for 1 h in PBS with secondary antibodies: Alexa Fluor 488 donkey anti-chicken, Cy5 donkey anti-chicken, Alexa Fluor 594 donkey anti-rabbit, and/or Cy5 donkey anti-rabbit (1:500, Jackson ImmunoResearch). The tissue was washed 3 times in PBS, mounted onto glass slides, and coverslipped with Fluoromount-G (Southern Biotech). Fluorescent images were acquired using a confocal microscope. All digital images were processed in the same way between experimental conditions to avoid artificial manipulation between different datasets.

Proper targeting of the MD and vHPC was verified for all mice used for electrophysiology. After mPFC slices were prepared, the remaining brain tissue was postfixed by immersion fixation in 4% paraformaldehyde at 4° for at least 48 h. The tissue was then washed for 24 h in phosphate buffered saline (PBS) before 100 μm slices through the MD and vHPC were prepared on a vibratome (Leica). Immunostaining to detect EYFP, GFP, or mCherry was performed using the following primary antibodies: EYFP or GFP (chicken-anti-GFP; Abcam; ab13970, 1:1000), mCherry (rabbit-anti-dsRed; Takara Bio; 632 496, 1:500); tdTomato was visible without additional amplification. Alexa Fluor-conjugated secondary antibodies (Invitrogen, 1:1000) were used for secondary detection.

Coronal MOE sections (14 μm thickness) from P14 mice were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. The sections were washed with PBS three times (5 min each) and incubated with permeable buffer (0.3% Triton X-100 in PBS) containing 5% donkey serum for 30 min. Primary antibodies against TAAR4, TAAR5, TAAR6 (homemade), caspase-3 (Cell Signaling, 9661), GFP (Abcam, ab13970), and tdTomato (Takara, 632496) were used at 1:5000, 1:5000, 1:1000, 1:500, 1:1000, and 1:500 dilution, respectively, in incubation buffer (1% bovine serum albumin, 0.01% sodium azide, 0.3% Triton X-100 in PBS). Primary antibody incubations were performed at 4 °C for two overnights. The sections were then rinsed three times (5 min each) in PBS and incubated at 37 °C for 30 min with different fluorophore-conjugated secondary antibodies (diluted at 1:1000), including Donkey Anti-Chicken IgY conjugated to Alexa Fluo 488 (Jackson ImmunoResearch, 703-545-155), Donkey Anti-Rabbit IgG conjugated to Alexa Fluo 488 (Jackson ImmunoResearch, 711-545-152), and Donkey Anti-Guinea Pig IgG conjugated to Cy3 (Jackson ImmunoResearch, 706-165-148). Slides were rinsed three times (5 min each) and coverslipped using mounting medium containing DAPI (SouthernBiotech). Images were taken using a Leica TCS SP8 confocal microscope.