Abcam Ad

Author Topic: will staining pattern affect?  (Read 7029 times)

0 Members and 1 Guest are viewing this topic.

Offline apple

  • GoldMember
  • *****
  • Posts: 150
will staining pattern affect?
« on: January 30, 2006, 12:11:07 PM »
Hi,

I used both ICC and IF stained some cytospin slides. I found very interested results. ICC show the protein expression pattern is nuclei staining. But IF staining show the protein expression pattern is membrane staining. Is it possible? It sounds very unlikely.

Thanks for your suggestion

will staining pattern affect?
« on: January 30, 2006, 12:11:07 PM »

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
will staining pattern affect?
« Reply #1 on: January 30, 2006, 11:05:13 PM »
There are potentially different levels of sensitivity in one method vs another and it is true that slightly different staining patterns may sometimes be observed; however, completely opposite staining patterns seriously sounds like some sort of background to me.

Do you have pictures? It would certainly help in this case.
[/color]

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
will staining pattern affect?
« Reply #2 on: January 30, 2006, 11:45:26 PM »
Sometimes the staining diffuses to surrounding area. The pattern may look different from that it is supposed to be. What is the antibody supposed to stain? Nuclei or Membrane?

Pictures will help.

Richard

Offline apple

  • GoldMember
  • *****
  • Posts: 150
will staining pattern affect?
« Reply #3 on: January 31, 2006, 06:16:48 PM »
Hi, the antigen I am looking for is Oct-3/4. I read some reference and this protein is suppose to be nuclei staining pattern. I do have some pictures and how could I show the pictures to you? Many thanks for help.

I did at least 15 cytospins. 10 with IF method and 5 with ICC method. ICC works fine. I can see clear nuclei staining pattern on sample slides. And no positive signal on control slides. But for IF staining, it shows membrane staining pattern and I can see positive signals on both sample and control slides. I am confused now. How to explain this?  If the antibody isn't specific binding, why did ICC work fine?

Thanks

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
will staining pattern affect?
« Reply #4 on: January 31, 2006, 06:50:19 PM »
To post pictures, go to Image Gallery at http://www.ihcworld.com/imagegallery/, register, then check your email and follow the link to activate your registration. Now you can login and upload images. Note: the image size limit is 100MB so make sure your images are smaller than 100MB. JPG file is prefered.

Quote from: "apple"
Hi, I do have some pictures and how could I show the pictures to you? Many thanks for help.

Offline apple

  • GoldMember
  • *****
  • Posts: 150
will staining pattern affect?
« Reply #5 on: February 01, 2006, 12:35:48 PM »
Hi,

I have upload some pictures at Fluorescent label folder. Oct-3/4 is a transcription factor. Mose paper say it is expressed in nuclei. My results show it's expression pattern is cytoplasmic.

Thanks

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
will staining pattern affect?
« Reply #6 on: February 01, 2006, 11:36:40 PM »
It looks like "peri-nuclei" although some of the staining is diffused into cytoplasmic area. So I would still consider it as nuclear staining pattern.

Quote from: "apple"
Hi,

I have upload some pictures at Fluorescent label folder. Oct-3/4 is a transcription factor. Mose paper say it is expressed in nuclei. My results show it's expression pattern is cytoplasmic.

Thanks

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
will staining pattern affect?
« Reply #7 on: February 02, 2006, 12:24:59 AM »
I respectfully but strongly disagree ... it does not look nuclear at all to me but cytoplasmic! There is no overlap with PI at all and perinuclear does not equal nuclear!! (Although I don't even think it is perinuclear, it appears solidly cytoplasmic.) It would also help if you had a phase pic to see the cell outlines.[/color]

Quote from: "richard03"
It looks like "peri-nuclei" although some of the staining is diffused into cytoplasmic area. So I would still consider it as nuclear staining pattern.

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
will staining pattern affect?
« Reply #8 on: February 02, 2006, 12:26:18 AM »
Incidentally where did you buy this antibody? This antigen is famously tricky to work out so be careful of false positive results.[/color]

Offline apple

  • GoldMember
  • *****
  • Posts: 150
will staining pattern affect?
« Reply #9 on: February 02, 2006, 05:06:24 AM »
Hi MaximinaNYC and richard03,

I bought the antibody from Santa Cruz. It's monoclonal mouse anti human ab (SC-5279). Why do you think this is a famous tricky antigen?

I also did ICC on the same cells with the same ab and same protocol. ICC results show clear cytoplasmic staining (maybe because nuclear pattern is difficult to tell from ICC). And negative control is completely negative. So I am lost now. I don't know if my cells are positive or negative. I will upload some ICC photoes today. Please have a look.
I will also upload some phase contrast photoes.

Many many thanks for help

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
will staining pattern affect?
« Reply #10 on: February 02, 2006, 07:30:07 AM »
On a second look, I have to agree with you that majority of the staining is indeed cytoplasmic staining pattern although I do see some cells appear to be "nuclear like" pattern. Some ICC pictures with cell outline will clearify this even more.
 
Quote from: "MaximinaNYC"
I respectfully but strongly disagree ... it does not look nuclear at all to me but cytoplasmic! There is no overlap with PI at all and perinuclear does not equal nuclear!! (Although I don't even think it is perinuclear, it appears solidly cytoplasmic.) It would also help if you had a phase pic to see the cell outlines.[/color]

Quote from: "richard03"
It looks like "peri-nuclei" although some of the staining is diffused into cytoplasmic area. So I would still consider it as nuclear staining pattern.

Offline apple

  • GoldMember
  • *****
  • Posts: 150
will staining pattern affect?
« Reply #11 on: February 02, 2006, 08:21:56 PM »
I just uploaded some ICC photoes. I think my ICC staining works well. Samples show both positive color (dark brown) and blue nuclear color. I think it is nuclear staining pattern.
Contorl only shows blue nuclei color. Do you agree with me?

Offline apple

  • GoldMember
  • *****
  • Posts: 150
will staining pattern affect?
« Reply #12 on: February 02, 2006, 08:23:25 PM »
my photoes are in Immunohistochemistry (enzyme labeled method) folder.

Thanks

will staining pattern affect?
« Reply #12 on: February 02, 2006, 08:23:25 PM »