Tissues were processed into paraffin, sections cut on a Leica RM 2135
rotary microtome and dried overnight at 36°C on Fisher Superfrost Plus
slides. Sections were deparaffinized and rehydrated, and antigen retrieval
performed (120°C, 3 minutes) using DIVA Decloaker (Biocare Medical).
After washing in distilled water twice for 3 minutes, sections were blocked in
PBS containing 5% normal goat serum, 1% bovine serum albumin, and 0.25% triton
X-100 in a humidified chamber for 1 hour at RT, then incubated in primary
antibody GALNS (Epitomics) 1:50, normal rabbit IgG in block solution or Collagen
X (Santa Cruz Biotechnology) 1:100 for 2 hours at RT. After 3 washes for 5
minutes in PBS, sections were incubated in goat anti-rabbit IgG Rhodamine Red-X
(Invitrogen) 1:300 or Alexa Fluor 488 (Jackson Immunoresearch) in block solution
for 1 hour at RT in the dark, washed 3 times for 5 minutes in PBS in the dark,
rinsed in distilled water, and coverslipped in Prolong Gold with DAPI
(Invitrogen).
rotary microtome and dried overnight at 36°C on Fisher Superfrost Plus
slides. Sections were deparaffinized and rehydrated, and antigen retrieval
performed (120°C, 3 minutes) using DIVA Decloaker (Biocare Medical).
After washing in distilled water twice for 3 minutes, sections were blocked in
PBS containing 5% normal goat serum, 1% bovine serum albumin, and 0.25% triton
X-100 in a humidified chamber for 1 hour at RT, then incubated in primary
antibody GALNS (Epitomics) 1:50, normal rabbit IgG in block solution or Collagen
X (Santa Cruz Biotechnology) 1:100 for 2 hours at RT. After 3 washes for 5
minutes in PBS, sections were incubated in goat anti-rabbit IgG Rhodamine Red-X
(Invitrogen) 1:300 or Alexa Fluor 488 (Jackson Immunoresearch) in block solution
for 1 hour at RT in the dark, washed 3 times for 5 minutes in PBS in the dark,
rinsed in distilled water, and coverslipped in Prolong Gold with DAPI
(Invitrogen).