Rabbit polyclonal anti-Glcci1 was obtained from Sigma-Aldrich (HPA001674) and used at 1:200 dilution for IHC and IF. Citrate retrieval was necessary to detect the Glcci1 signal. Boiling citrate retrieval buffer was added to slides in a coplin jar and covered for 30–40 minutes at room temperature. IHC and IF were carried out using standard techniques as described previously (Waclaw et al., 2010 (link)). A tyramide amplification kit (Invitrogen-ThermoFisher Scientific) was used to detect the fluorescent Glcci1 signal. Goat polyclonal anti-Sp8 (Santa Cruz, 1:3000) and Guinea-Pig anti-Ascl1 (kind gift from J. Johnson at UT-Southwestern, 1:10,000) were used for fluorescent double stains with the rabbit anti-Glcci1 antibody. Secondary antibodies were purchased from Jackson Immunoresearch (Cy3 or Cy5 conjugated Donkey-anti-Goat or Cy3 conjugated Donkey-anti-Guinea-Pig used at 1:200 dilution). Bright-field images were taken on a Leica DM2500 upright microscope with Leica DFC-500 camera using Leica acquisition software (Leica Microsytems). Fluorescent images were taken on a Nikon C2-Confocal Microscope using Nikon Elements software (Nikon Instruments Inc.).