Immunofluorescence Staining of Collagen VI

Colon sections were from previous animal studies using an AOM-induced CRC model in A/J mice [10 (link)]. Methcarn-fixed paraffin embedded colon sections were deparaffinized with xylene and rehydrated in an ethanol gradient. The slides were incubated in a citrate buffer at 95°C for 15 minutes, cooled to room temperature (RT), rinsed with PBS and incubated in a blocking buffer (PBS containing 1% Saponin and 20% BSA) for 30 minutes. The slides were then incubated with rabbit anti-ColVI (1:200, Abcam) at 4°C overnight, washed with PBS, and incubated with donkey-anti-rabbit Alexa 594 for 1 hour at RT. The slides were washed again, stained with DAPI, mounted and examined in a DeltaVision Elite microscope (GE Healthcare).