Re: [Histonet] positive controls - adequacy?

From:"Diane G. Miller"

Hi Satoshi,

I  work in cell culture and we are using magnetic beads to pull out endothelium cells from the buffy coats of 50 ml blood samples. If you need to know the vendor for the beads, please contact me directly, it was pricey.  I stained for CD34 and CD31, the CD 31 is  positive, but I'm still having problems getting the right antibody for the CD34. There are internal controls that we know should  stain.  I have a very nice image of the membrane stain on the CD31 if you would like me to send it to you. It also depends on the passage of the cells, whether you are going to get positive staining or not.  I have been very happy with the staining I've done with a new polymer kit that's just come out called PolyDetector by Bio SB.  It has eliminated any background staining that I've experienced with other kits, especially since I'm working with Baboon blood. 

I would think you could use cells from the Buffy coat as a control.  Just transfer some cells to 4 well chamber slides and feed with appropriate media.  Grow to  at least 80% confluency. Then fix with either 4% paraformaldehyde or 10% Buffered formalin for no more than 10 minutes, rinse in TBS,  store in 70% alcohol until ready to stain. Then proceed with staining.

If I can assist you with any protocols please let me know. 

Best Regards
Diane G. Miller, HT(ASCP), QIHC
Senior Research Associate
  ----- Original Message ----- 
  From: Satoshi Akima 
  Sent: Thursday, March 17, 2005 4:17 AM
  Subject: [Histonet] positive controls - adequacy?

  Dear Listmembers,

  this is my first post on the list and I was hoping to canvass some 

  I am doing some research and would like to stain for neutrophils, 
  lymphocytes and macrophages (to see which cell type seems to have a 
  pathogenic role). The question is what is good enough as a positive 
  control, especially when you are using commercial antibodies for which 
  a published body of literature supporting the specificity of the 
  antibody is out there. Would you:

  A. Just use a blood smear (whole blood)
  B. Use white cells from the Buffy coat
  C. Use a column (like the miniMACS system using antibody couples 
  magnetic beads) to separate out lineages
  D. In the case of mononuclear cells use Lymphoprep to separate out PBMCs
  E. Culture your PBMCs to get a purer isolate of macrophages

  Option C is nice but would cause a big time blow out in the budget - 
  all just to get a positive control for IHC. Seems a bit over the top. 
  I'm also in a hurry to get my paper out and time management is an issue 
  (reagents take time to order in from overseas).

  What do people think?
  Thanks for your help.

  Toshi Akima
  PhD Student
  Centre for Transplantation and Renal Research
  Westmead Millenium Institute
  Sydney, Australia

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