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Anti-TLR9 antibody (ab37154)

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1 question for ab37154

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Question 1

Monday 10-October-2011

Dear

Sir, madam,

Please can you give me more information about your antibody

Anti-TLR9 antibody (ab37154)

We like to use it in mouse paraffin section, and fixed in formalin.

 

My question is about antigen retrieval system (EDTA or Sodium citrate or Prot K).

How long and in microwave or boiling in pressure cooker.

 

Greeti

ANSWER:

  

Thank you for contacting Abcam about ab37154.

Please find below the suggested standard IHC-P protocol recommended for this antibody, however you may need to optimize it for your own specific needs.

Standard Protocol:

BUFFERS

Working Citrate Buffer

9mL of 0.1M Citric Acid (10.5g citric acid monohydrate to 500mL DI H2O)

41mL of 0.1M Sodium Citrate (14.7g sodium citrate dehydrate to 500mL DI H2O)

450mL of DI H2O

Deparaffinization and Rehydration (Cover staining dishes with a lid in each step)

1. Dip slides in three (3) changes of xylene or a xylene substitute for 3 minutes each.

2. Dip slides in two (2) change of 100% alcohol for 3 minutes each.

3. Dip slides in one (1) change of 95% alcohol for 3 minutes.

4. Dip slides in one (1) change of 70% alcohol for 3 minutes.

5. Rinse slides twice (2x) in DI H2O for 5 minutes.

Antigen Retrieval (Microwave Method)

6. Soak slides in 3% H2O2 for 5 minutes.

7. Rinse slides twice (2x) in DI H2O for 5 minutes.

8. Soak the slides in the working citrate buffer and cover with a lid.

9. Microwave until the liquid boils, about 1-5 minutes.

10. Remove from heat and let it stand at room temperature for 20 minutes.

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11. Wash three (3) times for 5 minutes in DI H2O

12. Remove the liquid (do not touch the tissue!) and use a PAP pen to circle around the tissue.

Blocking

13. Apply enough 5% BSA with a transfer pipette to cover the tissues.

14. Incubate the slides overnight at 4°C in a humid chamber.

Primary Antibody

15. Dilute the primary antibody to the recommended concentration in 1% BSA/PBS diluent.

16. Remove the BSA, and incubate with primary antibody solution for 1 hour at room temperature.

17. Wash slides three (3) times 5 minutes each on the shaker.

Secondary Antibody and Detection

18. Dilute the biotinylated secondary antibody to 1:200 in 1% BSA diluent.

19. Incubate with secondary antibody solution for 30 minutes at room temperature.

20. Wash slides in PBS three (3) times 5 minutes each on the shaker.

21. Add enough streptavidin HRP to cover the tissues. Incubate for 30 minutes at room temperature

22. Wash three (3) times 5 minutes each in PBS on the shaker.

23. Add enough DAB to cover the tissues. Once the cells start turning brown (inexperienced

technicians may wish to observe this under a microscope), wash twice (2x) in PBS for 5 minutes

each on the shaker.

If there is anything else I can help

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