Goat anti chicken af488

Immunostaining on formalin-fixed paraffin embedded rat mammary tissue was performed as previously described [94 (link)]. The following primary antibodies were used in this study: chicken anti-KRT5 (Biolegend, San Diego, California, USA; 905903, Lot# B279859, 1:1000), mouse anti-E-cadherin (BD Biosciences; 610182, Lot# 7292525, 1:400) and rabbit IBA1 (FUJIFILM Wako Chemicals; 019–19741, Lot# CAK1997, 1:1000). The following secondary antibodies (Thermofisher Scientific) were used in this study (1:500): goat anti-chicken AF488 (A11039), goat anti-mouse AF555 (A32727) and goat anti-rabbit AF647 (A21245). Nuclei were stained with DAPI dilactate (625 ng/ml) for 10 min. Tissue was imaged using an Olympus BX63F upright epifluorescence microscope. Brightness and contrast was optimally adjusted for lineage markers and DAPI. Exposure, brightness and contrast were kept consistent between tissue sections for IBA1.

After fixation and washes in PBS/Triton-X (PBT) 0.5%, brains were blocked overnight in PBS/Triton-X (PBT) 0.5% supplemented with Bovine Serum Albumin (BSA) 1% and then incubated with the following antibodies: α-Imp (rabbit, 1:1000; Medioni et al., 2014 (link)), α-Imp (rat, 1:1000; Medioni et al., 2014 (link)), α-Me31B (rabbit, 1:3000; Lee et al., 2017 (link)), α-Me31B (mouse, 1:3000; Nakamura et al., 2001 (link)) α-pCamkII (rabbit, 1:1000; Santa Cruz Biotechnology, sc-12886-R), α-GFP (chicken, 1:1000; Abcam, #ab13970). After incubation with primary antibodies, brains were washed three times with PBT 0.5% and incubated overnight with secondary antibodies. The following secondary antibodies were used in this study: Goat anti-rat AF568 (Thermofisher, A-11077), Goat anti-rat AF647 (Thermofisher, A-21247) Donkey anti-rabbit AF568 (Thermofisher, A-10042), Donkey anti-rabbit AF647 (Thermofisher, A-31573), Donkey anti-mouse AF488 (Thermofisher, A-21202), Donkey anti-mouse AF568 (Thermofisher, A-10037), Donkey anti-mouse AF647 (Thermofisher, A-31571), and Goat-anti-chicken AF488 (Thermofisher, A-11039). DAPI was used at 5 μg/mL and incubated for 5 min after secondary antibody incubation. Brains were washed in PBT 0.5% three times following secondary antibody incubation and were then mounted in vectashield (Vector Laboratories).

Mice were anaesthetized and perfused with 4% PFA in PBS. Heads were post-fixed, decalcified, cryoprotected, frozen and sectioned at 12 µm, as described [40 (link)]. IHC was performed as described [40 (link)]. The following antibodies were used: chicken-anti-βGal (Abcam, ab9361), chicken-anti-GFP (Abcam, ab13970), rabbit-anti-Dsred (Clontech, 632496), donkey-anti-rabbit AF555 (Invitrogen, A31572), goat-anti-chicken AF488 (Invitrogen, A11039). Multi-colour ISH was performed as previously described [67 (link)]. For riboprobes that were used, see the electronic supplementary material, table S1. For quantifying the percentage of OSNs at distinct maturation states in figure 5a,b, every 10th section was collected from anterior to posterior and 42 sections were analysed per mouse.