GFP Immunohistochemistry on Mouse - Paraffin

GFP Immunohistochemistry on Mouse - Paraffin

GFP Antibody - Molecular probes rabbit polyclonal, cat# A11122 Note: works best on lung, variable on other tissues

  1. Heat tissues to 60°C for 10 mins
  2. 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. 5% ethanol, 5 min d. 70% ethanol, 5 min

  3. Rinse the slides once in dH20

  4. Wash with PBS for 5 mins

  5. Block: Quench endogenous peroxidase using 3% H202 in methanol for 20 mins @RT

  6. Wash PBS 2 x 5 min

  7. Block: incubate in PBS/1%BSA for 20 mins at 37°C

  8. Wash PBS 2 x 5 min

  9. Primary Antibody: dilute anti-GFP (Mol Probes cat# A11122) or isotype control 1:100 in PBS/1%BSA, incubate 1 hour at 37°C

  10. Wash PBS 3 x 5 min

  11. Secondary Antibody: dilute anti-rabbit HRP (Dako cat#PO448) 1:100 in PBS/1%BSA and incubate for 30 min at 37°C

  12. Wash PBS 5 x 5 min

  13. Develop: use DAB or other peroxidase substrate

Diane Krause, MD, PhD
Professor of Laboratory Medicine, of Cell Biology and of Pathology; Assoc. Director, Yale Stem Cell Center; Assoc. Director, Transfusion Medicine Service; Medical Director, Clinical Cell Processing Laboratory; Medical Director, Advanced Cell Therapy Laboratory