Alexa fluor 647 anti rabbit

CG, NG, DRG, brain, spleens and livers were immersed in 30% sucrose (Sigma-Aldrich, 50389) in PBS overnight, then embedded in O.C.T compound (ThermoFisher, 23–730-572), frozen at −80 °C and sectioned at 10 μm thickness. Tissues were stained overnight with mCherry (Abcam, ab205402) + synapsin1 (Cell Signaling, 5297S) or mCherry + tyrosine hydroxylase (TH, Millipore, AB152) at 1:1,000 dilution. Subsequent secondary antibodies used were Alexa Fluor 647 anti-rabbit (Jackson ImmunoResearch, 711–605-152) and Alexa Fluor 594 anti-chicken (Jackson ImmunoResearch, 703–585-155). Tissues were stained with DAPI and coverslipped as stated previously. Samples were visualized using a fluorescent Zeiss Axio Observer Z.1 microscope.
Image analysis was performed using FIJI. To assess mCherry+ expression in brain, liver, spleen, GC, NG and DRG, images were converted to Tiff format, thresholding applied then made binary. The watershed function was applied to separate particles and cell quantification performed using the ‘analyze particle’ function. Results are shown as percentage of positive cells (mCherry+) in the total population (DAPI) or total number of positive cells. The quantification of mCherry+ overlap with synapsin+ fibres in the liver was done using the JaCOP plugin in FIJI.

To detect general anesthesia-activated neurons, animals were anesthetized with isoflurane, or ketamine/xylazine, or dexmedetomidine, for two hours then transcardially perfused with 10% sucrose in cold phosphate buffer saline (PBS, pH 7.4) followed by 4% cold paraformaldehyde (PFA) fixation solution. To detect restraint stress-activated neurons, mice were placed in the restrainer for 90 min (TV-150, Braintree scientific Inc). All brains were post-fixed in PFA overnight at 4°C, cryoprotected in 30% sucrose PBS solution for 2–3 days at 4°C, frozen in O.C.T compound (Tissue-Tek, Sakura), and then stored at −80°C until sectioning. Floating brain sections (80 µm) were stained using standard immunofluorescent protocol. The primary antibody used were: goat anti-Fos (Santa Cruz Biotechnology, sc520g, 1:300)⁵⁰ , rabbit anti-Fos (Cell Signaling, #2250, 1:1500)⁵¹ , anti-Neurotensin (ImmunoStar, 20072)⁵² . The secondary antibody used are: Alexa Fluor 488 donkey anti-goat (Jackson ImmunoResearch, 705-545-147, 1:500)⁵³ , Alexa Fluor 647 anti-rabbit (Jackson ImmunoResearch, 711-605-152, 1:500)⁵⁴ , and Cy3 donkey anti-goat (Jackson ImmunoResearch, 705-165-147, 1:500)⁵⁵ (see Life Sciences Reporting Summary).

Animals were anesthetized and transcardially exsanguinated with PBS (pH 7.4) and perfused using 10% formalin (30 mL). Brains were post-fixed overnight in 10% formalin and placed in PBS at 4°C before sectioning. Cerebella were sectioned into 50 m sagittal sections using a vibratome. Epitopes were exposed using antigen retrieval by boiling sections four times for 10 sec each in 0.01M urea. Sections were blocked overnight in 2% normal donkey serum and 0.3% Triton X-100 in PBS. Subsequent staining was carried out in 2% normal donkey serum and 0.3% Triton X-100 in PBS. Anti-calbindin antibodies used were (Sigmmouse a-Aldrich Cat# C9848/RRID:AB-10115846) and rabbit (Sigma-Aldrich Cat# C2724/RRID:AB-258818) at a 1:250 dilution.
Sections were incubated for 24 hrs with primary antibodies at 4°C. Following incubation, sections were washed three times in PBS and exposed to secondary antibodies (Alexa Fluor 488 antimuse-cat# 715-546/RRID:AB-2340850 and Alexa Fluor 647 antirabbit-cat# 711-605-152/RRID:AB-2492288}, Jackson Immunoresearch Labs, West Grove, PA) for 24 hrs at 4°C. Sections were washed three times in PBS and mounted onto charged slides (Colorfrost Plus, Fisher, Waltham, MA). Fluorescently labeled tissue was imaged using a confocal Olympus 1000 IX inverted microscope.