Abcam Ad

Author Topic: high background (IF)  (Read 4781 times)

0 Members and 1 Guest are viewing this topic.

Offline khairunnisa

  • Trainee
  • **
  • Posts: 9
high background (IF)
« on: October 13, 2010, 03:18:36 AM »
I'm using rat anti-mouse primary antibody, detecting neutrophil on mouse tissue. Even without primary antibody, I can see high background even after diluting the secondary antibody further. How do I omit the high background? The secondary antibody I'm using is goat-anti-rat, Alexa Fluor 555. Could it be that the secondary antibody cross react with my mouse tissue? How do I overcome it???

high background (IF)
« on: October 13, 2010, 03:18:36 AM »

Offline MT Scientist

  • GoldMember
  • *****
  • Posts: 116
Re: high background (IF)
« Reply #1 on: October 21, 2010, 12:14:11 PM »
You will need to post your detailed protocol before any assessments can be made.

Offline khairunnisa

  • Trainee
  • **
  • Posts: 9
Re: high background (IF)
« Reply #2 on: November 01, 2010, 02:55:24 AM »
Hi, the detailed protocol are as below.

(1) Fix the tissue in 2%PFA for 10 mins
(2) wash 3x in PBS for 5min
(3) Permeablize the tissue in 0.1% Triton X-100 for 5 mins
(4) Block the tissue with 5%BSA for 1hour
(5) Incubate primary antibody(in this case rat anti-mouse for neutrophil) overnight.
(6) wash 3x in 0.1%PBST for 5 min each
(7) Incubate secondary antibody 1hr
(8) Wash 3 x in 0.1%PBST for 5 min each
(9) Counterstain with DAPI

Offline MT Scientist

  • GoldMember
  • *****
  • Posts: 116
Re: high background (IF)
« Reply #3 on: November 03, 2010, 02:16:50 PM »
The background could be due autofluorescence from the tissue itself after fixation in PFA.  People have used many methods to quench autofluorescence (Na borohydride, sudan black, etc) and excellent resources are available from the internet (sorry I can't be more specific, but they were fairly easy to find)

What does your negative control look like (primary omitted)?  Will answer whether your secondary is binding non-specifically.

Does unstained tissue show the fluorescence (replace primary and secondary with buffer).  This will show you whether it is your tissue that is autofluorescing (see above).

Have you titrated your primary antibody?  Too much primary antibody can lead to autofluorescence.

« Last Edit: November 03, 2010, 02:23:16 PM by MT Scientist »

Offline khairunnisa

  • Trainee
  • **
  • Posts: 9
Re: high background (IF)
« Reply #4 on: November 03, 2010, 11:31:21 PM »
My negative control is without primary antibody, when I add secondary, there is still background.

My secondary antibody is goat anti-rat and I'm using mouse aorta tissue, therefore could it be that the secondary bind non-specifically to my tissue?

For aorta I know that the elastin will autofluorescence but the medial, adventitia area showed backround even if I use the most diluted secondary antibody alone.


I've use different dilution of the primary antibody and even I use the most diluted secondary antibody, there is still background.



Offline Cardio

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 249
Re: high background (IF)
« Reply #5 on: November 18, 2010, 05:14:47 PM »
Its time to buy a better secondary and get your money back.


Re: high background (IF)
« Reply #5 on: November 18, 2010, 05:14:47 PM »