GFP Antibody - Clontech mouse monoclonal, clone JL-8 cat# 8371-2
Prepare MOM solutions according to manufacturers instructions- Vector MOM Peroxidase kit (cat# PK-2200) or MOM basic kit (cat# BMK-2202). All incubations take place at room temperature unless indicated.
- Heat tissues to 60°C for 10 mins
- Deparaffinize and rehydrate tissue sections through series of xylenes and ethanol incubations a. Xylenes or alternative (Citrisolve, Histoclear) incubate 2 times, 5 min each incubation (2 x 5 min) b. 100% ethanol, 5 min c. 95% ethanol, 5 min d. 70% ethanol, 5 min
- Rinse the slides once in dH20
- Wash with PBS for 5 mins
- Antigen Retrieval: Preheat BD retrievagen A solution (BD Pharmigen cat# 550524) in coplin jar in steamer. Add slides to coplin jar and incubate for 20 min in steamer then let stand at room temperature for 30 min.
- Wash with PBS 2 x 5 min
- Block: Incubate slides in PBS/3% BSA/0.05%Tween20 for 30 min at 37°C.
- Wash in PBS 2 x 5 min
- Primary Antibody: Dilute anti-CD45 1:20 (Santa Cruz cat# sc-18846) or isotype in MOM diluent, incubate for 1 HR at RT
- Wash PBS 2 x 5 min
- Secondary Antibody: anti-rat-alexa 647 (Mol Probes) 1:100 in PBS and incubate for 1 HR at RT
- Wash PBS 2 x 5 min
- Block: biotin block using Vector kit (cat# SP-2001)
- Wash PBS 2 x 5 min
- Block: MOM block, 30 min at 37°C
- Wash PBS 2 x 5 min
- Diluent, tip off
- Primary Antibody: Dilute anti-GFP 1:250 or isotype in MOM diluent and incubate for 1HR at RT
- Wash PBS 2 x 5 min
- Secondary Antibody: Incubate in MOM biotinylated reagent for 10 min at RT
- Wash PBS 2 x 5 min
- Tertiary Antibody: Dilute Streptavidin-alexa 488 (Mol Probes) 1:500 in PBS/1%BSA and incubate for 30 min at 37°C
- Wash PBS 2 x 5 min
- Antigen Retrieval: 0.5% 1x trypsin (Gibco cat#16438, 0.25% trypsin, 1mM EDTA) preheated to 37°C, incubate 30-35 seconds
- Wash PBS 2 x 5 min
- Primary Antibody: Dilute anti-pankeratin (DAKO cat#Z0622) 1:500 in MOM diluent incubate ON at 4°C
- Wash PBS 2 x 5 min
- Secondary Antibody: Dilute anti-rabbit-alexa 568 (Mol Probes) 1:100 in PBS/1%BSA and incubate for 30 min at 37°C Wash and view
