Anti-GDNF Receptor alpha 1 antibody (ab53079)

We have a customer who has purchased the above antibody and would like to know which protocol was used for the image on the datasheet. See her query below;

I purchased an Abcam antibody recently (Rabbit Anti-GDNF Receptor alpha 1 Polyclonal Antibody, Unconjugated, cat#ab53079) and according to the Abcam website, it has been used successfully for IHC on paraffin embedded sections (they also show a picture). However they do not indicate the protocol they used to get this result. Is there a way to find the exact protocol that was used so that I can trial it on my samples? Their website has a PDF file of IHC protocols, but it states all the different protocols that can be used (eg Antigen retrieval) but not the method that was used to obtain the results they show in the picture. I wait for your response.

ANSWER:

Thank you for your enquiry.

Please find the protocol used for this product below.

Deparaffinization

1. Incubate slide at 60℃ for 60 minutes.

2. Deparaffinize in Xylene for 10 minutes and repeat one more times.

3. Hydrate in 100% alcohol for 5 minutes, in 95% alcohol for 5 minutes, in 85% alcohol for 5 minutes, in 75% alcohol for 5 minutes.

4. Dip into Distill Water for 5 minutes.

5. Dip into TBS (50 mM Tris, 100 mM NaCl, pH 7.6), leave for 5 minutes, and repeat two times.

Antigen Retrieval

6. Bring 500 - 2000 ml 10 mM citrate buffer (pH6.0) to the boil in a stainless steel pressure cooker.

7. Put the slide into staining rack and lower into pressure cooker ensuring the slide is well immersed in citrate buffer.

8. When the pressure indicator valve has risen after 3-4 minutes, incubate for 1 minute.

9. Cool the slide naturally to room temperature.

10. Dip into distilled water, leave for 5 minutes, and repeat two times.

11. Dip the slide in TBS for 5 minutes and repeat two times.

12. Immerse slides in 3% H2O2 ( in fresh methanol) for 15 minutes at room temperature.

13. Wash with distilled water two times, 5 minutes each time.

14. Wash with TBS (pH 7.6) two times, 5 minutes each time.

Staining with Primary Antibody

15. Dilute primary antibody with 3% BSA in TBS. Cover the tissue section on the slide with diluted primary antibody (use 50 – 150μl for each slide).

16. Incubate at 37℃ for 30 minutes or at room temperature for 60 minutes (The optimal incubation time, incubation temperature, and antibody dilution should be determined by the individual laboratory).

17. Wash with TBS two times, 5 minutes each time.

Staining with Secondary Antibody

18. Incubate with 100-200μl Polymer Enhancer. Incubate 30 minutes at 37℃.

19. Wash with TBS for 3 times, 5 minutes each time.

20. Incubate with 100-200μl Polymerized HRP and incubate 30 minutes at 37℃

21. Wash with TBS for 3 times, 5 minutes each time.

22. Add DAB solution and incubate 3-10 minutes(The reaction progress and the optimal time should be determine according to microscope).

23. Wash with distilled water for 2 times, 5 minutes each time.

24. Counterstain sections in hematoxylin if required,wash with distilled water.Immerse slides in 0.1% HCl- ethanol for 1-10 seconds, wash with distilled water.

25. Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2×3min, Xylene for 2×3min, and coverslip with mounting medium.

I hope this information will be helpful.