Donkey anti chicken igg irdye 680lt

DAPI (dilution 1:1000, Thermo Fisher Scientific, 62248), Goat anti-Hamster IgG Alexa Fluor 647 (dilution 1:200, Jackson ImmunoResearch, 127-605-160), Goat anti-Rabbit IgG Alexa Fluor 430 (dilution 1:200, Thermo Fisher Scientific, A11064), Goat anti-Rabbit IgG Alexa Fluor 430 (dilution 1:200, Thermo Fisher Scientific, A11064), Goat anti-Rabbit IgG Alexa Fluor 594 (dilution 1:200, Thermo Fisher Scientific, A11037), Goat anti-Mouse IgG1 Alexa Fluor 488 (dilution 1:200, Thermo Fisher Scientific, A21121), Goat anti-Mouse IgG1 Alexa Fluor 647 (dilution 1:200, Thermo Fisher Scientific, A21240), Goat anti-Mouse IgG2a Alexa Fluor 546 (dilution 1:200, Thermo Fisher Scientific, A21133), Goat anti-Mouse IgG2b Alexa Fluor 647 (dilution 1:200, Thermo Fisher Scientific, A21242), Goat anti-Guinea Pig IgG Alexa Fluor 546 (dilution 1:200, Thermo Fisher Scientific, A11074), Goat anti-Guinea Pig IgG Alexa Fluor 594 (dilution 1:200, Thermo Fisher Scientific, A11076), Donkey anti-Chicken IgG IRDye 680LT (dilution 1:200, Li-Cor Biosciences, 926-68028), Donkey anti-Rabbit IgG IRDye 800CW (dilution 1:200, Li-Cor Biosciences, 926-32213), Donkey anti-Chicken IgG Alexa Fluor 488 (dilution 1:500, Jackson ImmuoResearch, 703-545-155), and Donkey anti-Guinea Pig IgG Alexa Fluor 647 (dilution 1:500, Jackson ImmuoResearch, 706-605-148).

Mice were anesthetized with ketamine/xylazine and transcardially perfused with PBS. The brain and spinal cords were quickly harvested. The brain and spinal cords were dounced using a tissue douncer with RIPA buffer (pH 7.5, 25 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) and the appropriate dilution of protease and phosphatase inhibitors. The dounced tissue was then sonicated and stored at −80 °C. Protein levels were normalized using a Bradford Assay and were assessed with Li-Cor Odyssey CLx infrared imaging system. Primary antibodies used for western blot were used as follows: goat anti-CCL21-unconjugated (1:100; AF457-SP; R&D Systems), rabbit anti-CCR7-unconjugated (1:100; ab32527; Abcam), rabbit anti-VEGFC-unconjugated (1:100; ab83905; Abcam), and chicken anti-ß-actin-unconjugated (1:1000; ab13822; Abcam). The appropriate secondary antibodies were used as follows: donkey anti-rabbit IgG-IRDye 800CW (1:2000; 926–32213; Li-Cor), donkey anti-goat IgG-IRDye 800CW (1:2000; 925–32214; Li-Cor), and donkey anti-chicken IgG-IRDye 680LT (1:2000; 926-68028; Li-Cor). For a detailed list of reagents, refer to Supplementary Table 1. Representative uncropped western blots for VEGFC and ß-actin can be found in Supplementary Fig. 11.