Ab150133

The catalytic domain of Arabidopsis HMGR1S (CD1) produced in Escherichia coli was used as immunogen to produce a polyclonal antibody in rabbit and the resulting serum was immunosubstracted to remove IgG reacting against the bacterial proteins [48 (link)]. The immunopurified serum (Ab-CD1-i) was used as primary antibody at 1:500 for whole mount and 1:1000 for transmission EM. Anti-rabbit IgG secondary antibodies for HMGR detection were code Ab150066 (Abcam, Toronto, ON, Canada) coupled to Alexa Fluor-555 at 1:1000 for whole mount (Figure 1a,b), code Ab150068 (Abcam, Toronto, ON, Canada) coupled to Alexa Fluor-594 at 1:1000 for whole mount (Figure 1g), code 111-215-144 (Jackson Immunoresearch, Cambridge, UK) coupled to an 18-nm gold particle at 1:30 for transmission EM (Figure 1d) and code 111-205-144 (Jackson Immunoresearch, Cambridge, UK) coupled to a 12-nm gold particle at 1:30 for transmission EM (Figure 1e,f,l,m).
GFP was detected with Ab-5450 (Abcam, Toronto, ON, Canada) as the primary antibody at 1:1000 for whole mount and transmission EM. Anti-goat IgG secondary antibodies for GFP detection were code Ab150133 (Abcam, Toronto, ON, Canada) coupled to Alexa Fluor-488 at 1:1000 for whole mount, and code 705-215-147 (Jackson Immunoresearch, Cambridge, UK) coupled with an 18-nm gold particle at 1:15 for transmission EM.

Shrews (n = 5–6 shrews per group) were treated with either vehicle or yohimbine (1 mg/kg, i.p.) and rapidly anesthetized with isoflurane and subjected to perfusion at 15 min and 30 min post-treatment to examine 5-HT and SP immunoreactivity. The experimental procedure prior to staining was performed as described above for Section 4.4.1. c-fos Staining and Image Analysis. Coronal brainstem sections (20 μm) were blocked with 0.1 M PBS containing 10% donkey serum and 0.3% Triton X-100, then incubated overnight at 4 °C with a mix of goat anti-5-HT primary antibody (1:1000, ab66047, Abcam) and rat anti-SP primary antibody (1:400, MAB356, EMD Millipore, Burlington, VT, USA) in 0.1 M PBS containing 5% donkey serum and 0.3% Triton X-100. Sections were washed 3 times (10 min each) in PBS and incubated in a mix of Alexa Fluor 488 donkey anti-goat (1:500, ab150133, Abcam) and cy3-conjugated donkey anti-rat (1:500, AP189C, EMD Millipore) secondary antibody in 0.1 M PBS containing 0.3% Triton X-100 for 2 h at room temperature. After washing with PBS 3 times (10 min each), sections were mounted with anti-fade mounting medium containing DAPI (Vector Laboratories). Images for the DVC were acquired using a confocal microscope (Zeiss LMS 880) as described above. Fluorescence intensity (mean gray value) of 5-HT and SP values were acquired using ImageJ software, as described previously [45 (link)].

LC3B and p62 protein expression levels were measured by western blotting. Hippocampal and prefrontal cortex tissues (n = 5) of rats were washed, homogenized, and lysed in RIPA buffer (MA0151, Meilunbio) containing protease inhibitors. After routine protein extraction from brain tissues, the total protein concentration was determined, and then 30 μg of total protein was mixed with 1/4 volume of 5× SDS‐PAGE sample buffer (P0015, Beyotime Biotechnology). The protein samples were boiled for 10 min, and the proteins were separated by SDS‐PAGE. After transferring the separated proteins onto PVDF membranes (IPVH 00010, Millipore), the membranes were incubated with primary antibodies, such as LC3B (1:1000, ab192890, Abcam), p62 (1:1000, 23214S, CST), and GAPDH (1:5000, 97166, CST), at 4°C overnight. Subsequently, secondary antibodies (1:5000, ab150133, Abcam) were incubated at 37°C for 2 h. The absorbance values of the protein bands were scanned using an Odyssey infrared gel imaging system (Affinity, USA). Quantitative analysis was performed using ImageJ software (version 1.45 J; National Institutes of Health, Bethesda, MD, USA).