Tgf-beta1 Ihc Protocol

TGF-β1 Immunohistochemistry Investigators: G. Huang, L. Zuckerwise Note: sections farther from label are neg controls (no primary Ab) TGF-β1 Ab: Abcam, Ab27969

Put paraffin embedded slides in a 60oC oven for ~ 1 hour. Deparaffinize slide through: 1. Xylene (2x10min) 2. EtOH/xylene, 1:1 (3min) 3. EtOH 100% (2x3 min) 4. EtOH 95% (3 min) 5. EtOH 70% (3 min) 6. EtOH 50% (3 min) 7. Rinse in cold tap water 8. TBS x 5 min Antigen Retrieval 1. Heat large beaker of water to 95 oC & add slide container w/ antigen retrieval solution to beaker to also heat the antigen retrieval solution 2. Add the slides to the antigen retrieval solution and leave in 95 oC for 20 min 3. Let the slides cool in TBS x 5min 4. Dry around sections and draw circular perimeter w/ hydrophobic pen 5. Incubate 20min with peroxidase block from Dako kit (2 drops/cut) Blocking 1. Block slides in 5% donkey serum and 2% BSA in TBS: o 10 mL TBS o 0.5 mL goat serum o 0.2 g BSA fraction 2. Block the slides for 60 min at room temperature 3. Remove blocking sol’n by draining slides and wiping around sections Staining Day 1 1. Create Primary Antibody solution (total volume 200 uL per slide): 2. Apply primary Ab 3. Incubate overnight at 4°C. Day 2 1. Tap off antibody & wash 4x5mins in TBS-Tween (0.1%) on shaker. Leave in TBS. 2. Tap off & add Secondary Rabbit polymer-HRP antibody (DAKO Envision+ Kit) x 30-45min.

3. Tap off & wash with TBS-Tween for 2x5min. Leave in TBS. 4. Prepare DAB: a. 20 uL per 1 mL of buffer b. 100 uL per section 5. Monitor reaction: 30s-1min. Stop reaction in TBS bath. 6. Wash with running tap water x 3 min. 7. Counterstain w/ prepared Hematoxylin (filtered; 1:3) x 15 sec. Stop reaction in TBS bath. 8. Wash with water until clear. 9. Pass quickly through NH3-water bath (50ul in 400ml beaker with deionized water. 10. Pass reverse through alcohol baths and xylene baths a. 50% EtOH x 3 min b. 75 EtOH x 3min c. 95% EtOH x 3min d. 100% EtOH 2x3 min e. EtOH/xylene, 1:1 x 3 min f. Xylene 2x 10 min; then leave in bath of clean xylene 11. Add 2 drops of DPX mountant b/t cuts and carefully place coverglass 12. Leave to dry overnight