Hi Nichakarn. Without knowing more detail it is difficult to know for certain, but uneven staining in otherwise same tissue from same source on the same slide, would indicate to me that availability of your target epitope is uneven. What antigen retrieval method did you use?

Hi Veda Deliv, I used citrate buffer for antigen retrieval. Today I‘m trying to do free floating method. I’m not sure which way good for GFAP staining (astrocyte in spinal cord).

Thank you

Hi Nichakarn,

I have only used the free floating method for staining GFAP, but it always worked very well (for PFA-fixed brain slices). What is the fixation method and thickness of your slices? What is the composition of the solution that you dilute your primary antibody in (e.g. 3% serum and 0.3% Tx-100 usually work very well)?

In cases where I had problems with uneven stainings or weak signals, longer incubation times (up to 5 nights) often solved the problem.

Good luck!

Hi Nichakarn,

Recently after deparaffinizing sections I tried several methods myself to improve CD 68 staining. For paraffin sections I found that ( 0.01 M Tris-1 mM EDTA, pH 9) worked better than sodium citrate a more gentle antigen retrieval. I even put my paraffin sections into a pressure cooker for 10 minutes which increased the signal. Hope this help.

Hi Vedad Delic, Thank you so much for your suggestions

I will try to do this. 🙂

Hi Henrike Horn, Thank you for your comments

Today I just got a good result when I changed to use floating method for GFAP staining . Also I used 3% serum and 0.3 tx-100 for dilute primary antibody. I think that floating method it’s good for me.

Thank you