1. Protein Blocking step is too short. Try 30-60 min. What is in the blocking solution?

2. Can you please send me the company and catalog number info of the antibody?

3. What primary dilutions did you try?

3. I never change the dilution of the chromagen... only the staining time. I use DAB or other HRP permanent chromagens.

4. Dehydrate the slides after chromagen, wash with distilled water and place slides through 70%, 95%, 100% graded alcohols , xylol or xylene and mount with a xylene based mounting medium (DPX, Permount, Cytoseal, etc.)

I would be happy to help, please contact me at [email protected]

Thank you,

Tina

Dear Colleague,

Immunohistochemical staining of bone samples is difficult.

The most important step on which the result depends is bone decalcification. How long and with what chemical did you carry out decalcification? If you used trichloro-acetic acid, then in most cases you will get a negative result, or a very weakly positive. It is best to soak pieces of bone tissue for half an hour in a decalcification solution to soften it a bit, and then in formalin for at least 24 hours.

For the rest, I prefer the retrieval antigen in pH 9.0 buffer (Dako) for 20 min at 97-98oC, and primary antibody overnight. You can also try a higher concentration of the primary (less diluted). I use hydrogen peroxidase blocking for 5 minutes, and protein blocking depending on the primary antibody (mostly I don’t use it).

Chromogenic staining is a little too long. In addition, do not change 3x5min, but leave the same DAB solution 1x10 min. (background reduction).

Good luck…

Hi

Snjezana Ramic Thank you for your answer! We established an organotiypic bone model where we use femur of 4 old day rat pups. We than cultivate them for up to 15 days and process them into 5µm FFPE slices. I was told that because of the young age (epiphysis is still cartilage) that decalcification is not neccessary.

I will try to use a HIER buffer with pH 9.0 as you recommend and block for longer and try some DAB chromogen for development.

Thank you for your help, I really appreciate it!

Hi

Montina Van Meter Thank you for your answer. I wrote you an Email with all the details. Thanks again!

Dear Vanessa,

I noticed that you use a Decloacking Chamber (under pressure or not?).

I use water bath for 20 minutes, but when I used decloacking chamber under pressure antigen retrieval had to be very short (like, two minutes). For a longer time under pressure you will lose the reaction.

Ensure that deparaffinization and re-hydration is performed well.

Best regards,

Snjezana