Biotinylated donkey anti goat igg

Two hours after the final presentation of the paired stimulus animals were deeply anesthetized with isoflurane and euthanized via transcardial perfusion. After post-fixation and cryoprotection, brains were coronally sectioned (44 um) on a freezing microtome. All tissue for immunohistochemical analysis was processed according to previously described protocols with minor modifications [17 (link),18 (link)]. Representative sections through the rostrocaudal extent of the brain were first mounted and stained for CTb to verify retrograde tracer deposit placement. Free-floating sections were incubated in goat anti-CTb antibody (1:7500; List Biological Laboratories) for 48 hr at 4 °C followed by biotinylated donkey anti-goat IgG (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1.5 hr at room temperature, and horseradish peroxidase-conjugated streptavidin (1:1600; Jackson ImmunoResearch Laboratories Inc.) for 1 hr at room temperature. Staining was visualized by addition of 3 % hydrogen peroxide to sections incubating in diaminobenzidine, yielding brown CTb-immunopositive labeling.

For immunohistochemistry, paraffin-embedded, bouin-fixed sections were deparaffinized, rehydrated and boiled in citrate buffer for 15 min. After protein blocking step, sections with antibodies for p-smad2/3 (SC-11769R, Santa Cruz Biotech.), myeloperoxidase (PA5-16672, Thermo Fisher Sci.), CD3 (A0452, Dako) and cleaved caspase-3 (#9664, Cell signaling Tech.) were applied to sections for 2 h. Slides were rinsed in PBS and incubated in either biotinylated donkey anti-goat IgG (#705-065-147, Jackson Immunoresearch Labs.) or biotinylated donkey anti-rabbit IgG (#711-065-152, Jackson Immunoresearch Labs.). For F4/80 and α–SMA immunostaining, Frozen-sections were stained with anti-α–SMA (M085, Dako) and F4/80 antibody (MCA497G, AbD Serotec) followed by incubation with Alexa fluor 594 goat anti-mouse IgG_2a (invitrogen) or biotinylated donkey anti-rat IgG (#712-065-153, Jackson Immunoresearch Labs). Immunoreactive cells were scored in ten high-power fields (20x) per mouse. At least five mice from control and mutant groups were analyzed in each immunostaining.