Abcam Ad

Author Topic: Does someone work with granulocytes ?  (Read 6750 times)

0 Members and 1 Guest are viewing this topic.

Offline fregata

  • Trainee
  • **
  • Posts: 8
Does someone work with granulocytes ?
« on: March 11, 2006, 04:00:46 AM »
Hi!!
 I stain cytospin preparation of granulocytes.
My protocol is:
FIx absoloute metanol 5'
BSA 3% 30'
Primary AB 1 H 37C
Secondary AB 1H 37C

I observe an aspecific staining of secondary antibodie (one made in donkey and one made in chicken): in the negative control (no primary antibody) the secondary antibodies bind aspecifically the citoplasm (the nucleos are clean).
I tried to diluite the secondary antibody form 1/500 to 1/2000 but results don't change and I changed fixing method (Formaldeyde 4% and the Tritox 0.015) with the same results.

Now could the saturation with the serum of donkey and chicken eliminate the aspecific?

does someone have some advices ?

Thanks much

   Gianluca

Does someone work with granulocytes ?
« on: March 11, 2006, 04:00:46 AM »

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
Does someone work with granulocytes ?
« Reply #1 on: March 11, 2006, 06:09:02 PM »
That's might be autofluorescence. I assume you have used fluorescent detection method.

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
Re: Does someone work with granulocytes ?
« Reply #2 on: March 13, 2006, 12:58:24 AM »
Try blocking with serum.
Try to do your antibody incubations at RT (no need for 37 deg C!!)
Try to fix with acetone instead of MeOH.
What fluorophore are you using for detection?
[/color]
Quote from: "fregata"
I stain cytospin preparation of granulocytes.
My protocol is:
FIx absoloute metanol 5'
BSA 3% 30'
Primary AB 1 H 37C
Secondary AB 1H 37C

I observe an aspecific staining of secondary antibodie (one made in donkey and one made in chicken): in the negative control (no primary antibody) the secondary antibodies bind aspecifically the citoplasm (the nucleos are clean).
I tried to diluite the secondary antibody form 1/500 to 1/2000 but results don't change and I changed fixing method (Formaldeyde 4% and the Tritox 0.015) with the same results.

Now could the saturation with the serum of donkey and chicken eliminate the aspecific?

Offline fregata

  • Trainee
  • **
  • Posts: 8
Does someone work with granulocytes ?
« Reply #3 on: March 13, 2006, 05:17:34 AM »
I use alexa donkey anti-rabbit 546.
I observed only a weak autofluorescence, very weak, so the signal observed in the negative control is done by aspecific secondary binding!!!
I've yet used serum to block aspecific bindind, I will try with it.

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
Does someone work with granulocytes ?
« Reply #4 on: March 20, 2006, 02:01:01 PM »
Is your secondary adsorbed against various species including the one you are staining? This would also help.[/color]

Quote from: "fregata"
I use alexa donkey anti-rabbit 546.
I observed only a weak autofluorescence, very weak, so the signal observed in the negative control is done by aspecific secondary binding!!!
I've yet used serum to block aspecific bindind, I will try with it.

Offline kishore

  • Newbie
  • *
  • Posts: 3
Re; problem with auto fluroscence
« Reply #5 on: October 29, 2010, 09:31:35 AM »
Hi..
I am working with RAT RETINAL sections, the problem is getting signal in control also.
so i have a little dought about autofluroscence, how can i check that, some of my friends saying that should not dry the sections[working with parafin sections], so i tried with 75% glycerol/.01 M PBS for mounting the section for auto fluorescence, Is it right way to check, IS their any method other than this.   

Offline excalibur

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 1109
    • http://www.excaliburpathology.com
Re: Does someone work with granulocytes ?
« Reply #6 on: October 29, 2010, 02:48:39 PM »
How are you preparing the sections?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline kishore

  • Newbie
  • *
  • Posts: 3
Re: Re; problem with auto fluroscence
« Reply #7 on: November 09, 2010, 05:17:33 AM »
Hi
after dissection of eye balls immediately fixed in 4% para formaldehyde,then next day paraffin blocks preparation.
I am working with this paraffin retinal rat sections for immunofluroscence.My primary Ab is mouse anti-rhodapsin[monoclonal]
secondary Ab is anti-rabbit alexa fluor-555. problem is getting noise to signal ratio is very high. help me in this issue

Offline kishore

  • Newbie
  • *
  • Posts: 3
Re: Re; problem with auto fluroscence
« Reply #8 on: November 13, 2010, 04:17:25 AM »
HI
Can anyone tell the  quenching procedure for PFA sections

Re: Re; problem with auto fluroscence
« Reply #8 on: November 13, 2010, 04:17:25 AM »