Abcam Ad

Author Topic: nuclear staining: high background  (Read 2854 times)

0 Members and 1 Guest are viewing this topic.

Offline yasiangel

  • Newbie
  • *
  • Posts: 3
nuclear staining: high background
« on: February 23, 2007, 12:13:38 PM »
The specimens are rat spinal cord cut with the cryostat machine 10 ul thick.
 
Here is the protocol I am using:
 
1. Rinse slides in PBS to rehydrate 5 min 3 times
2. blocking with 8% goat serum & 0.4% Triton X solution for 1 hour
3. Rinse with 1 quick PBS wash and slides allowed to air dry
4. Specimens circled with PAP pen and primary Ab added
 
1:100 HuNu (chemicon MAB 1281)
1:200 Mitchondrial Antibody (mouse)
1:100   "                 "
1:10     "                "
 
5. Refrigrated overnight
6. Afterwards, excess solution tapped off and washed with PBS 3 times 5 min each
7. Secondary Ab added (flourescence)
 
Hoechst solution 1:1000
goat anti-mouse secondaey Ab 1:500
 
8. incubate in humidity chamber @ room temp 2 hours
9. Rinse once quick in PBS, then rinse again 3 times @ 5 min each
10.  Add gel to slide and put on cover slip and store overnight in dark
 
The secondary anitbodies are labeled with flourecense, but when i look at them under the microscope there is a lot of background staining all over the slide.
 
what can I do to reduce the background staining?
 
 
thank you,

nuclear staining: high background
« on: February 23, 2007, 12:13:38 PM »

Offline excalibur

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 1109
    • http://www.excaliburpathology.com
Re: nuclear staining: high background
« Reply #1 on: February 24, 2007, 05:00:14 PM »
You do not state what fixation you are using.

Also, do not dry the sections in step 3 after blocking.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
Re: nuclear staining: high background
« Reply #2 on: February 28, 2007, 12:26:23 AM »
Saying you are using dilutions such as 1:1000 and 1:500 mean nothing without knowing the concentration. So what are the concentrations of primaries and secondaries you are using. Also and importantly, is your anti-mouse IgG secondary cross adsorbed against rat IgG? Rat and mouse IgG are so similar that if you do not use an antibody that is highly serum adsorbed to all species including rat you will get high background.

Offline yasiangel

  • Newbie
  • *
  • Posts: 3
Re: nuclear staining: high background
« Reply #3 on: March 01, 2007, 05:51:47 PM »
Here are images from the slides.  Samples are fixed in agarose (4% paraformaldehyde for 1h) and then cut after freezing in OCT.

HuNu 1:100


1:200 Mitch Ab


1:100 Mitch Ab


1:10 Mitch Ab



Primary Antibodies:

1:100 HuNu (chemicon MAB 1281)
1:200 Mitochondrial Antibody (mouse)
1:100   Mitochondrial Ab (mouse)
1:10     Mitochondrial Ab (mouse)



Fluorescent labeled secondary Ab used is Alexa-flouro 546 (red) (concentration 2 mg /ul), but also 488 (green) was used and the same problem occurred with high background.  The secondary ab anti-mouse IgG secondary is cross adsorbed against rat IgG.  We have not carried out a dilution series with these antibodies but will try that next.


Question: How would skipping the drying step help? Do I dry the slides prior to adding the cover slip?

Also, although specific signals are seen, not all of the double staining co-localize as seen in the images.

Re: nuclear staining: high background
« Reply #3 on: March 01, 2007, 05:51:47 PM »