Alexa488 goat anti chicken

Following primary antibodies were used for Immunofluorescence (IF) and Western blotting (WB): rabbit anti-GFP (1:200 IF, Invitrogen, A11122), chicken anti-GFP (1:200 IF, Aves, GFP-1020), rabbit anti-GFAP (1:100 IF, Dako, Z0334), mouse anti-NeuN (1:50 IF, Millipore, MAB377), rat anti-L1 (1:100 IF, Millipore, MAB5272), rabbit anti-Calretinin (1:500 IF, Millipore, MAB5054), goat anti-Nrp1 (2 µg/ml for function blocking, R and D, AF566), goat anti-VEGFR2 (1:15 IF, R and D, AF644), rabbit anti-VEGFR2 (1:1000 WB, Cell Signaling, 2479), rabbit anti-(phospho)VEGFR2 (Y1175) (1:500 WB, Cell Signaling, 2478), rabbit anti-(phospho/SFK (Y416) (1:200 IF, Invitrogen, 44660G), mouse anti-beta-III-tubulin (1:150 IF, Sigma, T5076), goat anti-VE-Cadherin (1:1000 WB, R and D, AF1002), Phalloidin (1:400 IF, Sigma, P1951). The following secondary antibodies were used: donkey anti-rabbit Alexa488 (Jackson Immunoresearch, 711-545-152), donkey anti-rabbit Alexa568 (Life Technologies, A10042), donkey anti-goat Alexa568 (Molecular Probes, A11057), goat anti-mouse Alexa568 (Invitrogen, A11031), goat anti-rat Alexa568 (Invitrogen, A11077), goat anti-chicken Alexa488 (Molecular Probes, A11039), donkey anti-goat HRP (Jackson Immunoresearch, 705–0350147), donkey anti-rabbit HRP (Jackson Immunoresearch, 711-035-152), donkey anti-mouse HRP (Jackson Immunoresearch, 715-035-150).

Mice were perfused, and brains were postfixed for 24 hr in 10% formalin. Brain slices were taken using a vibratome (Leica, Buffalo Grove, IL), blocked for 1 hr with 0.3% Triton X-100, 3% bovine serum albumin (BSA), and 2% normal goat serum (NGS) and incubated in primary antibodies for 24 hr at 4°C. Then, free-floating slices were washed three times for 10 min in 0.1% Triton X-100 in PBS (PBS-T), incubated for 1 hr at room temperature with secondary antibodies, washed in PBS-T and mounted in Vectamount with DAPI (Southern Biotech, Birmingham, AL). Antibodies used here were: anti-c-fos (1:500; Cell Signaling, Danvers, MA), anti-mCherry (1:1000; Abcam, Cambridge, MA) goat-anti-rabbit (Alexa 488 or Alexa 594, 1:1000; Molecular Probes), goat anti-chicken Alexa488 or Alexa594 (1:1000; Molecular Probes). Images were taken using Axiovert 200 microscope (Zeiss, White Plains, NY) or LSM780 confocal (Zeiss) and images were processed using ImageJ software (NIH, Schneider et al., 2012 (link)). C-fos counts were conducted for three or more sections/animal and averaged for each animal for statistical analysis.