Alexa fluor 568 donkey anti sheep igg

Manufactured by Thermo Fisher Scientific

Alexa Fluor 568 donkey anti-sheep IgG is a secondary antibody conjugated with the Alexa Fluor 568 fluorescent dye. It is designed to detect and visualize sheep immunoglobulin G (IgG) in various applications, such as immunohistochemistry, flow cytometry, and Western blotting.

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Alexa Fluor 568 (1190 citations) Alexa 568 (337 citations) Alexa Fluors (21 citations) Alexa Fluor 568 IgG (9 citations) Alexa Fluor 568 Donkey anti-Sheep (2 citations)

4 protocols using alexa fluor 568 donkey anti sheep igg

Immunofluorescence Analysis of Mouse Brain

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In all, 10- to 12-week-old mice (n = 7–8 per group) were perfused transcardially with 4% paraformaldehyde. The brains were then frozen, sectioned at 30-µm thick, and processed for immunofluorescence using standard procedures^{7 (link)}. The primary antibodies used for IHC were as follows: sheep anti-αMSH antibody (1:40,000, Millipore, cat #AB5087), rabbit anti-AgRP (1:1,000, Phoenix Pharmaceuticals, cat #H003-53), rabbit anti-LC3B (1:500, Cell Signaling, cat #2775), rabbit anti-p62 (1:1,000, Abcam, cat #ab64134), rabbit anti-GFP (1:1,000, Invitrogen, cat #A-6455), guinea pig anti-insulin (1:500, Abcam, cat #ab7842), and rabbit anti-vesicular acetylcholine transporter (VAChT, 1:500, Synaptic Systems, cat #139103). The primary antibodies were visualized with Alexa Fluor 568 donkey anti-sheep IgG (1:200, Invitrogen, cat #A21099), Alexa Fluor 488 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A21206), Alexa Fluor 488 donkey anti-mouse IgG (1:200, Invitrogen, cat #A21202), Alexa Fluor 568 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A10042), or Alexa Fluor 488 goat anti-guinea pig IgG (1:200, Invitrogen, cat #A11073). The sections were counterstained using bis-benzamide (1:10,000, Invitrogen, cat #H3569) to visualize cell nuclei.

Park S., Aintablian A., Coupe B, & Bouret S.G. (2020). The endoplasmic reticulum stress-autophagy pathway controls hypothalamic development and energy balance regulation in leptin-deficient neonates. Nature Communications, 11, 1914.

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Fluorescent Antibody Labeling Protocol

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Alexa Fluor 488 chicken anti-mouse immunoglobulin G (IgG) (Invitrogen #A21200, RRID_AB_2535786, used at 1:400); Alexa Fluor 488 chicken anti-rabbit IgG (Invitrogen #A21441, RRID_AB_10563745, used at 1:400); Alexa Fluor 568 donkey anti-sheep IgG (Invitrogen #A21099, RRID_AB_10055702, used at 1:400); Alexa Fluor Plus 488 goat anti-rabbit (ThermoFisher A32731, used at 1:500); Alexa Fluor Plus 594 goat anti-mouse (ThermoFisher A32742, used at 1:500); and goat anti-mouse IgG-HRP (horseradish peroxidase) (BioRad #170-6516, RRID:AB_11125547, used at 1:20000).

Gordon D.E., Hiatt J., Bouhaddou M., Rezelj V.V., Ulferts S., Braberg H., Jureka A.S., Obernier K., Guo J.Z., Batra J., Kaake R.M., Weckstein A.R., Owens T.W., Gupta M., Pourmal S., Titus E.W., Cakir M., Soucheray M., McGregor M., Cakir Z., Jang G., O’Meara M.J., Tummino T.A., Zhang Z., Foussard H., Rojc A., Zhou Y., Kuchenov D., Hüttenhain R., Xu J., Eckhardt M., Swaney D.L., Fabius J.M., Ummadi M., Tutuncuoglu B., Rathore U., Modak M., Haas P., Haas K.M., Naing Z.Z., Pulido E.H., Shi Y., Barrio-Hernandez I., Memon D., Petsalaki E., Dunham A., Marrero M.C., Burke D., Koh C., Vallet T., Silvas J.A., Azumaya C.M., Billesbølle C., Brilot A.F., Campbell M.G., Diallo A., Dickinson M.S., Diwanji D., Herrera N., Hoppe N., Kratochvil H.T., Liu Y., Merz G.E., Moritz M., Nguyen H.C., Nowotny C., Puchades C., Rizo A.N., Schulze-Gahmen U., Smith A.M., Sun M., Young I.D., Zhao J., Asarnow D., Biel J., Bowen A., Braxton J.R., Chen J., Chio C.M., Chio U.S., Deshpande I., Doan L., Faust B., Flores S., Jin M., Kim K., Lam V.L., Li F., Li J., Li Y.L., Li Y., Liu X., Lo M., Lopez K.E., Melo A.A., Moss FR I.I.I., Nguyen P., Paulino J., Pawar K.I., Peters J.K., Pospiech TH J.r., Safari M., Sangwan S., Schaefer K., Thomas P.V., Thwin A.C., Trenker R., Tse E., Tsui T.K., Wang F., Whitis N., Yu Z., Zhang K., Zhang Y., Zhou F., Saltzberg D., Hodder A.J., Shun-Shion A.S., Williams D.M., White K.M., Rosales R., Kehrer T., Miorin L., Moreno E., Patel A.H., Rihn S., Khalid M.M., Vallejo-Gracia A., Fozouni P., Simoneau C.R., Roth T.L., Wu D., Karim M.A., Ghoussaini M., Dunham I., Berardi F., Weigang S., Chazal M., Park J., Logue J., McGrath M., Weston S., Haupt R., Hastie C.J., Elliott M., Brown F., Burness K.A., Reid E., Dorward M., Johnson C., Wilkinson S.G., Geyer A., Giesel D.M., Baillie C., Raggett S., Leech H., Toth R., Goodman N., Keough K.C., Lind A.L., Klesh R.J., Hemphill K.R., Carlson-Stevermer J., Oki J., Holden K., Maures T., Pollard K.S., Sali A., Agard D.A., Cheng Y., Fraser J.S., Frost A., Jura N., Kortemme T., Manglik A., Southworth D.R., Stroud R.M., Alessi D.R., Davies P., Frieman M.B., Ideker T., Abate C., Jouvenet N., Kochs G., Shoichet B., Ott M., Palmarini M., Shokat K.M., García-Sastre A., Rassen J.A., Grosse R., Rosenberg O.S., Verba K.A., Basler C.F., Vignuzzi M., Peden A.A., Beltrao P, & Krogan N.J. (2020). Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms. Science (New York, N.y.), 370(6521), eabe9403.

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Immunofluorescence Staining of Hypothalamic Neuropeptides

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Sections were processed for immunofluorescence using standard procedures (Bouret et al., 2004b (link); Steculorum et al., 2015 (link); Croizier et al., 2016 (link)). The primary antibodies used for IHC were as follows: sheep anti-α-MSH (1:40,000, Millipore; Steculorum et al., 2015 (link)), sheep anti-NPY (1:3,000, Chemicon; Lee et al., 2013 (link)), rabbit anti-β-endorphin (1:5,000, Millipore; Grayson et al., 2010 (link)), rabbit anti-POMC (1:2,000, Phoenix Pharmaceuticals; MacKay et al., 2017 (link)). The primary antibodies were visualized with Alexa Fluor 568 goat anti-rabbit IgGs or Alexa Fluor 568 donkey anti-sheep IgGs (1:200, Invitrogen). Sections were counterstained using bisbenzamide (1:10,000, Invitrogen), to visualize cell nuclei, and coverslipped with buffered glycerol (pH 8.5).

Croizier S., Park S., Maillard J, & Bouret S.G. (2018). Central Dicer-miR-103/107 controls developmental switch of POMC progenitors into NPY neurons and impacts glucose homeostasis. eLife, 7, e40429.

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Immunofluorescence Analysis of Neuronal Markers

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Anesthetized male mice were perfused transcardially with 4% paraformaldehyde at 2–3 months of age. The brains were then frozen and sectioned at 30-μm-thick and processed for immunofluorescence using standard procedures. The primary antibodies used for immunohistochemistry (IHC) were as follows: rabbit anti-GFP (1:10,000, Invitrogen), rabbit anti-cFos (1:2,000, Oncogene), rabbit anti-p62/SQSTM1 (1:1,000, Abcam), and rabbit anti-ubiquitin (1,000, Dako). The primary antibodies were visualized with Alexa Fluor 488 or Alexa Fluor 568 goat anti-rabbit IgGs (1:200, Invitrogen) and Alexa Fluor 568 donkey anti-sheep IgGs (1:200, Invitrogen). Sections were counterstained using bis-benzamide (1:10,000, Invitrogen) to visualize cell nuclei and coverslipped with Fuoromount-G (SouthernBiotech).

Coupé B., Leloup C., Asiedu K., Maillard J., Pénicaud L., Horvath T.L, & Bouret S.G. (2021). Defective autophagy in Sf1 neurons perturbs the metabolic response to fasting and causes mitochondrial dysfunction. Molecular Metabolism, 47, 101186.

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