Rabbit anti rfp antibody

Western blots were probed using rabbit anti-FLAG antibody (Sigma, 1:2500), rabbit anti-RFP antibody (Rockland, 1:2500), or chick anti-GFP antibody (Aves Lab, 1:2000) in 4% milk in PBS-T (PBS and 0.1% Tween-20) for 1 hr. After washing three times (5 min each) in PBS-T, blots were incubated in HRP conjugated anti-rabbit IgG antibody (1:2500) or anti-chick IgY (1:2000) for 1 hr, washed for 5 × 5 min in PBS-T, and then incubated with Clarity ECL reagent (BioRad) for 5 min before being exposed for 1 min to HyBlot ES X-ray film (Denville Scientific) or ChemiDoc Imaging System (BioRad).

Immunofluorescence labelling was performed on free‐floating coronal brain sections. For kisspeptin and tdTomato immunostaining, sections were incubated overnight with a sheep anti‐kisspeptin‐10 antiserum (AC024, dilution 1 : 2000; gift from Alain Caraty) combined with a rabbit anti‐RFP antibody (dilution 1 : 2000; Rockland Immunochemicals, Pottstown, PA, USA) in TBS containing 2% normal donkey serum, 0.3% Triton‐X‐100 and 0.25% bovine serum albumin. After several washes with TBS, the sections were placed in biotinylated donkey anti‐sheep secondary immunoglobulins (dilution 1 : 200; Jackson ImmunoResearch, West Grove, PA, USA) and then incubated with a combination of Alexa Fluor 488‐conjugated strepavidin and Alexa Fluor 568‐conjugated goat anti‐rabbit immunoglobulins, each for 90 min at RT (dilution 1 : 200; Molecular Probes, Carlsbad, CA, USA). All sections were then washed, mounted on slides, air dried, and cover slipped with Vectashield Fluorescence Mounting Medium (Vector Laboratories, Inc., Burlingame, CA, USA). Controls consisted of the omission of primary and/or secondary antibodies for the different combinations, and these sections consistently failed to exhibit the appropriate immunofluorescence.