Goat anti rat igg h

Transverse sections (60 μm thick) of whole, debarked stems were treated with LM5 as described above, except incubations with MP-PBS and the antibodies were done at 30 °C and not at room temperature. Also the secondary antibody was goat anti-rat IgG (H + L chains) secondary antibody conjugated with alkaline phosphatase (0.2 mL; 1:100 dilution) (Invitrogen, New Zealand) in MP-PBS, and after the last PBS wash, the sections were treated for 15 min with substrate solution containing bromo-4-chloro-3-indolyl phosphate (BCIP), nitro-blue tetrazolium (NBT) and water (1:1:8 v/v) (BCIP/NBT substrate kit, Invitrogen). Alkaline phosphatase reacts with the substrate to produce an insoluble blue product. The sections were then washed in water (5×) before mounting in Aqua-Mount medium (Thermo Fisher, New Zealand). Control experiments were also done with the primary antibody omitted and the images recorded using a digital camera (see above).