Donkey anti goat igg hrp sc 2056

Bat-specific IgG was analysed via dot blot. Bat-mice were cheek bled post NP-KLH immunizations, sera samples were dotted onto nitrocellulose membrane (Pore size 0.45 µm; BIO-RAD, USA) and left to dry for 30 minutes. Blocking buffer (Tris-buffered saline with polysorbate 20 (TBS-T) and 5% low-fat milk powder) was used to block non-specific sites for 30 minutes at RT with agitation. After washing, membrane was incubated for 1 hour at RT with goat anti-bat IgG heavy and light chain antibody (A140-118B; Bethyl Laboratories, USA) in PBS-T containing 2.5% milk and for 1 hour with agitation, followed by 3 times 5 minutes wash with TBS-T. Secondary antibody incubation was done with donkey anti-goat IgG-HRP (SC-2056; Santa Cruz Biotechnology, USA) in PBS-T containing 2.5% milk for 1 hour at RT with agitation and subsequently washed with TBS-T for 3 times 5 minutes before reaction development using enhanced ECL reagent (Thermo Fisher Scientific, USA). Results were developed in the dark room for 10 seconds with CL-XPosure^TM Film (Thermo Fisher Scientific, USA).

Sera were collected before and after challenging with NP-KLH by cheek bleed. Bat immunoglobulin (Ig) content specific to NP-KLH antigens was assessed by ELISA. Briefly, microtiter plates (Thermo Fisher Scientific, USA) were coated with NP31-BSA (Biosearch Technologies, USA) at 10 µg/mL. Bat specific antibodies were detected with bat IgG heavy and light chain antibody (A140-118B; Bethyl Laboratories, USA), donkey anti-goat IgG-HRP (SC-2056; Santa Cruz Biotechnology, USA) and 3,3′,5,5′-tetramethylbenzidine (TMB) (Thermo Fisher Scientific, USA). Optical density (OD) was read at 450 nm with a plate reader (Tecan, USA).

Secondary antibodies include goat anti-rabbit IgG-HRP (sc-2054 [WB: 1:2000]), donkey anti-goat IgG-HRP (sc-2056 [WB: 1:2000]), and goat anti-mouse IgG-HRP (sc-2005 [WB: 1:2000]) were purchased from Santa Cruz Biotechnology. For IF, Alexa Fluor® 488 donkey anti-rabbit IgG (A21206), Alexa Fluor® 594 donkey anti-rabbit IgG (A21207), Alexa Fluor® 488 donkey anti-goat IgG (A11055), Alexa Fluor® 594 donkey anti-goat IgG (A11058), and Alexa Fluor® 594 donkey anti-mouse IgG (A21203) were all purchased from ThermoFisher Scientific®.