Vectastain elite abc kit standard and peroxidase substrate kit dab sk 4100

At the end of the experiment, animals were overdosed with pentobarbital and perfused using saline followed by either 4% paraformaldehyde (PFA) in 0.1M PO₄ buffer or a sequence of 1% PFA/1% glutaraldehyde/0.004% CaCl₂ and 1% PFA/1% glutaraldehyde/0.004% CaCl₂. Coronal sections of 70 μm thickness were made through the brainstem with a vibratome (Leica VT1200). Sections were processed with commercial kits (VECTASTAIN Elite ABC Kit (Standard) and Peroxidase substrate kit DAB SK-4100, Vector Laboratories, Inc. Burlingame, CA 94010) to visualize the biocytin. A background Nissl stain was performed using thionin 0.2%. The tissue was mounted, cleared and coverslipped. For each isolated MSO neuron, the rostrocaudal location of the cell body was expressed relative to the most rostral and most caudal poles of the nucleus, and the dorsoventral location was expressed relative to the dorsal and ventral border of the nucleus in the same slice as the cell body (Fig. 2b-e).