Methods and Techniques Discussion => Immunofluorescence (IF) => Topic started by: pex on August 10, 2005, 09:24:50 AM

Title: Immunofluorescence in bone sections?
Post by: pex on August 10, 2005, 09:24:50 AM
Hello, all!

I am doing immunofluorescence in bone paraffin sections, but the results are not so nice. some positive expressions in osteoblasts are very weak. but I have some good results for immunofluorescence in primary osteoblasts in vitro. How should I do it?

Any suggestions will be useful for me?
Thank you!

pex
Title: Immunofluorescence in bone sections?
Post by: richard03 on August 10, 2005, 09:48:17 AM
What is your protocol? Have you used any antigen retrieval method?
Title: Immunofluorescence in bone sections?
Post by: pex on August 10, 2005, 10:24:51 AM
Normal immunofluorescence protocol.

I use 0.1% trypsin ( 20min) for antigen retrieval.
I have already used different antibodies for immunosatining. One of them is working well, positive expressions can be found in cartilage, osteoblasts and osteocytes, but others only express in cartilage strongly.
Title: Immunofluorescence in bone sections?
Post by: richard03 on August 10, 2005, 10:48:29 AM
That's primary antibody problem.
Title: Immunofluorescence in bone sections?
Post by: pex on August 10, 2005, 10:55:33 AM
But other primary antibodies can express strongly in primary osteoblasts.
Title: Immunofluorescence in bone sections?
Post by: richard03 on August 10, 2005, 10:58:13 AM
What primary antibodies are you using? Are they exactly same clone?
Title: Immunofluorescence in bone sections?
Post by: pex on August 10, 2005, 11:01:42 AM
Rabbit anti-catalase

Rabbit anti-PMP70

Sheep anti-alkaline phosphatase

................
Title: Immunofluorescence in bone sections?
Post by: richard03 on August 10, 2005, 11:20:51 AM
You have three totally different antibodies. How can you expect they are working and expressing all the same?

My suggestion is to try different antigen retrieval methods.

Richard