Rabbit anti histamine

Fly head sections (10 μm thick) were prepared from adults that were frozen in OCT medium (Tissue-Tek, Torrance, CA). Immunolabeling was performed on cryosections sections with mouse anti-24B10 (1:100, DSHB), rat anti-LOVIT (1:100) (Xu and Wang, 2019 (link)), or anti-CSP (1:100, DSHB), rat anti-RFP (1:200, Chromotek, Germany), rabbit anti-Hdc (1:50), rabbit anti-GFP (1:200, Invitrogen, Carlsbad, CA), and rabbit anti-Ebony (1:200, lab of Dr S Carroll, University of Wisconsin, Madison, WI) as primary antibodies. For histamine immunolabeling, the rabbit anti-histamine (1:100, ImmunoStar, Hudson, WI) antibody was pre-adsorbed with carcinine, as previously reported (Xu et al., 2015 (link)). Goat anti-rabbit lgG conjugated to Alexa 488 (1:500, Invitrogen, Carlsbad, CA), goat anti-mouse lgG conjugated to Alexa 488 (1:500, Invitrogen, Carlsbad, CA), goat anti- rabbit lgG conjugated to Alexa 568 (1:500, Invitrogen, Carlsbad, CA), and goat anti-rat lgG conjugated to Alexa 647 (1:500, Invitrogen, Carlsbad, CA) were used as secondary antibodies. The images were recorded with a Zeiss 800 confocal microscope.

Histamine (2-(1H-imidazol-5-yl)ethanamine; dihydrochloride, ACROS Organics, Fair Lawn, NJ, USA), human red blood cells (RBCs, Interstate Blood Bank, Memphis, TN, USA), and serum (Interstate Blood Bank, Memphis, TN, USA), RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with HEPES, L-glutamine, hypoxanthine (Acros Organics), DL-lactic acid (Acros Organics), Hoechst 33342-trihydrochloride trihydrate (Invitrogen, Carlsbad, CA, USA), JC-1 (Invitrogen, Carlsbad, CA, USA), mercurochrome, Ringer’s solution (3 mM CaCl₂, 182 mM KCl, 46 mM NaCl, 10 mM Tris pH 7.2), 1-octen-3-ol (Sigma-Aldrich), and electrode gel (Parker Laboratories, Fairfield, NJ, USA). N-3-dimethylaminopropyl-N′-ethylcarbodiimide (Sigma-Aldrich, St. Louis, MO, USA, cat. #03449), paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA, cat. #15710), agarose (Sigma-Aldrich), goat serum (BSA; Jackson ImmunoResearch Laboratories, West Grove, PA, USA, cat. #001-000-162), rabbit anti-histamine (ImmunoStar, Hudson, WI, USA, cat. # 22939, RRID:AB_572245), Alexa Fluor 488 (Thermo Fisher, cat. #A-11008), and Vectashield^®PLUS (Vector Laboratories, Burlingame, CA, cat. #H-1900).

Fly heads were fixed with 4% paraformaldehyde for 2h at 4°C or 4% EDAC (for histamine staining), and immersed in 12% glucose overnight at 4°C. The heads were embedded in O.C.T^™ compound (Tissue-Tek, Torrance, USA), and 10μm thick cryosections were cut. Immunolabeling was performed on cryosections sections with mouse anti-24B10 (1:100, DSHB, http://dshb.biology.uiowa.edu/), rat anti-RFP (1:200, Chromotek, Martinsried, Germany), rabbit anti-Ebony (1:200, lab of Dr. S. Carroll, University of Wisconsin, Madison, USA), and anti-Tan (1:200, lab of Dr. B. Hovemann, Ruhr Universität Bochum, Germany) [30 (link)] as primary antibodies. For histamine staining, rabbit anti- histamine (1:100, ImmunoStar, USA) was used as a primary antibody. The antibody was preadsorbed with carcinine as previously reported [9 (link)]. Goat anti-rabbit lgG conjugated to Alexa 488 (1:500, Invitrogen, USA), goat anti-rat lgG conjugated to Alexa 568 (1:500, Invitrogen, USA) and goat anti-mouse lgG conjugated to Alexa 647 (1:500, Jackson ImmunoResearch, USA) were used as secondary antibodies. The images were recorded with a Nikon A1-R confocal microscope.