From: Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer
Pms2 | Ercc1 | Xpf | Ku86 |
---|---|---|---|
Cut 4 micron sections | Cut 4 micron sections | Cut 4 micron sections | Cut 4 micron sections |
Deparaffinize and rehydrate | Deparaffinize and rehydrate | Deparaffinize and rehydrate | Deparaffinize and rehydrate |
Antigen retrieval in microwave with VECTOR Antigen Unmasking Solution | Antigen retrieval in microwave with VECTOR Antigen Unmasking Solution | Antigen retrieval in microwave with VECTOR Antigen Unmasking Solution | Antigen retrieval in microwave with VECTOR Antigen Unmasking Solution |
Rinse with PBS and distilled water | Rinse with PBS and distilled H2O | Rinse with PBS and distilled water | Rinse with PBS and distilled water |
Incubate in 3% H2O2 in methanol for 20 min | Incubate in 3% H2O2 in methanol for 20 min | Incubate in 3% H2O2 in methanol for 20 min | Incubate in 3% H2O2 in methanol for 20 min |
Rinse in distilled water, PBS | Rinse in distilled water, PBS | Rinse in distilled water, TBST, PBS | Rinse in distilled water, PBS |
Place in Sequenza racks, rinse with PBS | Place in Sequenza racks, rinse with PBS | Place in Sequenza racks, rinse with PBS | Place in Sequenza racks, rinse with PBS |
Add 3 drops Background Sniper, 10 min at room temp | Add 3 drops Background Sniper, 10 min at room temp | Add 3 drops Background Sniper, 10 min at room temp | Add 1.5% normal goat serum in 2% BSA/PBS, 120 μl, one hour |
Rinse with TBST | Rinse with PBS | Rinse with TBST | Do Not Rinse |
Primary mouse monoclonal from Pharmingen at 10 μg/ml into 2% BSA/TBST, 120 μl/slide | Primary mouse monoclonal 8F1 from Neomarkers at 2 μg/ml with 2% BSA/PBS, 120 μl/slide | Primary rabbit polyclonal 3F2/3 from Abcam at 5 μg/ml in Renoir Red at 120 μl/slide | Primary mouse monoclonal H-300 from Santa Cruz at 2 μg/ml in 2% BSA/PBS, 120 μl/slide |
Incubate 2 hrs at room temp | Incubate 45 min at room temp | Incubate 1 hour at room temp | Incubate 45 min at room temp |
3 rinses with TBST | 3 rinses with PBS | 3 rinses with TBST | 3 rinses with PBS |
Polyclonal rabbit anit-mouse antibody Dako Biotinylated secondary antibody at 100 μl/slide at 1:100 dilution in 2% BSA in TBST | Polyclonal rabbit anit-mouse antibody Dako Biotinylated secondary antibody at 100 μl/slide at 1:300 dilution in 2% BSA/PBS | Mouse Probe (Biocare) 4 drops/slide | Goat anti-rabbit biotinylated IgG secondary antibody at 1:100 dilution in 2% BSA/PBS, 100 μl/slide |
Incubate 30 min at room temp | Incubate 30 min at room temp | Incubate 15 min room temp | Incubate 30 min at room temp |
3 rinses with TBST | 3 rinses with PBS | 3 rinses with TBST | 3 rinses with PBS |
Vectastain Elite avidin-biotin complex method kit 3 drops/slide | Vectastain Elite avidin-biotin complex method kit 3 drops/slide | Mouse Polymer (Biocare) 4 drops/slide | Vectastain Elite avidin-biotin complex method kit 3 drops/slide |
Incubate 30 min room temp | Incubate 30 min room temp | Incubate 15 min room temp | Incubate 30 min room temp |
2 rinses TBST, then PBS | 2 rinses PBS | 3 rinses TBST, then PBS | 2 rinses PBS |
Diaminobenzidine tetrachloride plus 0.04% H2O2 | Diaminobenzidine tetrachloride plus 0.04% H2O2 | Diaminobenzidine tetrachloride plus 0.04% H2O2 | Diaminobenzidine tetrachloride plus 0.04% H2O2 |
Counterstain with hematoxylin | Counterstain with hematoxylin | Counterstain with hematoxylin | Counterstain with hematoxylin |
Dehydrate with ethanols followed by xylene | Dehydrate with ethanols followed by xylene | Dehydrate with ethanols followed by xylene | Dehydrate with ethanols followed by xylene |
Mount with Cytoseal and coverslips | Mount w/Cytoseal and coverslips | Mount with Cytoseal and coverslips | Mount with Cytoseal and coverslips |