Protocol specific for GPR49 Antibody (NLS1235)

Immunohistochemistry

1. Prepare tissue with formalin fixation and by embedding it in paraffin wax.

2. Make 4-mm sections and place on pre-cleaned and charged microscope slides.

3. Heat in a tissue-drying oven for 45 minutes @ 60 degrees Celcius.

4. Deparaffinize the tissues by wash drying the slides in 3 changes of xylene 5 minutes each @ RT.

5. Rehydrate the tissues by washing the slides in 3 changes of 100% alcohol 3 minutes each @ RT.

6. Wash the slides in 2 changes of 95% alcohol 3 minutes each @ RT.

7. Wash the slides in 1 change of 80% alcohol 3 minutes @ RT.

8. Rinse the slides in gentle running distilled water 5 minutes @ RT.

9. Perform antigen retrieval by steaming the slides in 0.01M sodium citrate buffer (pH 6.0) @ 99-100 degrees Celcius for 20 minutes.

10. Remove the slides from the heat and let stand in buffer @ RT for 20 minutes.

11. Rinse the slides in 1X TBS-T for 1 minute @ RT.

**Do not allow the tissues to dry at any time during the staining procedure**

12. Begin the immunostaining by applying a universal protein block 20 minutes @ RT.

13. Drain protein block from the slides and apply the diluted primary antibody 45 minutes @ RT.

14. Rinse the slide in 1X TBS-T 1 minute @ RT.

15. Apply a biotinylated anti-rabbit IgG (H+L) secondary 30 minutes @ RT.

16. Rinse the slide in 1X TBS-T 1 minute @ RT.

17. Apply an alkaline phosphatase steptavidin 30 minutes @ RT.

18. Rinse the slide in 1X TBS-T 1 minute @ RT.

19. Apply an alkaline phosphatase chromagen substrate 30 minutes @ RT.

20. Rinse the slide in distilled water 1 minute @ RT.

**This method should only be used if the chromagen substrate is alcohol insoluble (ie: Vector Red, DAB)**

21. Dehydrate the tissue by washing the slides in 2 changes of 80% alcohol 1 minute each @ RT.

22. Wash the slides in 2 changes of 95% alcohol 1 minute each @ RT.

23. Wash the slides in 3 changes of 100% alcohol 1 minute each @ RT.

24. Wash the slides in 3 changes of xylene 1 minute each @ RT.

25. Apply cover slip.