Alexa fluor 488 conjugated anti guinea pig igg

Free-floating sections of the spinal cord were blocked for 1 h at room temperature with 5% BSA in PBST to block non-specific binding, then incubated for 48 h at 4°C with a mixture of rabbit polyclonal antibody against rat BNP (diluted 1:2000; Millipore) and either guinea-pig polyclonal CGRP antibody (1:200; T-5053, Peninsula Lab, San Carlos, CA, USA) or goat anti-ChAT antibody (diluted 1:500; Millipore). After three rinses with PBST, sections were incubated for 3 h at room temperature with the appropriate secondary antibodies. The secondary antibodies were Alexa Fluor 555-conjugated anti-rabbit IgG, Alexa Fluor 488-conjugated anti-guinea pig IgG, or Alexa Fluor 488-conjugated anti-goat IgG (diluted 1:500, Thermo Fisher Scientific Inc., Waltham, MA, USA). Negative control sections were subjected to the same procedures without primary antibody. After three washes with PBST, the sections were mounted on gelatin-coated glass slides and examined using a confocal laser-scanning microscope (LSM 510 META, Carl Zeiss, Heidelberg, Germany).