Abcam Ad

Author Topic: immunofluorescent astaining of CD68 on human adipose tissue  (Read 3186 times)

0 Members and 1 Guest are viewing this topic.

Offline advak

  • Newbie
  • *
  • Posts: 4
immunofluorescent astaining of CD68 on human adipose tissue
« on: August 07, 2007, 03:27:22 AM »
I am trying to stain CD68 on paraffin sections of human adipose tissue, but unfotunatly I get a non- specific staining, hence, that fat cells are also stained.
 I used both tris-EDTA and citrate buffer for antigen exposure.
my staining protocol was as follows:
1h blocking with 5% normal goat serum
1h 1:100 dako CD68 antibody
3*5' PBS wash
1h  1:50 CY2 goat anti mouse (jackson)
if anyone has an experience with immunofluorescent staining of CD68, or have any idea I would love to hear it.

immunofluorescent astaining of CD68 on human adipose tissue
« on: August 07, 2007, 03:27:22 AM »

Offline excalibur

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 1109
    • http://www.excaliburpathology.com
Re: immunofluorescent astaining of CD68 on human adipose tissue
« Reply #1 on: August 07, 2007, 09:25:15 AM »
All FFPE tissue will fluoresce some. No matter the antibody. It is caused by the formalin/PFA.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline CanuckPhD

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 296
Re: immunofluorescent astaining of CD68 on human adipose tissue
« Reply #2 on: August 08, 2007, 02:08:18 AM »
Is the background in your sections before you start the staining? If so then it is due to PFA/formalin. In this case try some of the background quenching protocols such as Sudan Black B before you do your staining.

If the background is present in your IgG control slide (negative control) then likely your Cy2 secondary is at too high of a concentration. Try diluting it out more, which may decrease your background. Alternatively try a ABC, DAB protocol for the antibody.

Good luck

Re: immunofluorescent astaining of CD68 on human adipose tissue
« Reply #2 on: August 08, 2007, 02:08:18 AM »