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I would like to do immunofluorescence staining and I need your advice before I could buy the antibodies: 1- I would like to have antibody against human PPAR-alpha. Have you monoclonal antibody? or can I use polyclonal antibody? What is your recommendation for the primary and the secondary antibody? 2- The cells are obtained from human bronchoalveolar lavage and the cells are human alveolar macrophages and alveolar T lymphocytes. 3- I would like to do double staining: PPAR-alpha as mentioned above and against human CD4 T lymphocytes. 4- The CD4 antibody should be against the intracellular domain of CD4. The antibody should be monoclonal. Or what do you think?. 5- Can you give a guarantee that the PPAR-alpha antibody will work? 6- It is worthwhile to mention that alveolar macrophages have a great potential for autofluorescence. What is your advice for maximal blocking and maximal blenching of the autofluorescence?
Asked on Oct 25 2011
Thank you for contacting us. The antibodies I recommend are: PPAR alpha rabbit polyclonal - ab61182 Secondary - goat anti-rabbit IgG Dylight 488-conjugated, pre-adsorbed ab96899 CD4 mouse monoclonal - ab25804 Secondary - rat anti-mouse IgG1 PE-conjugated, pre-adsorbed ab99605 The PPAR alpha antibody ab61182 is guaranteed for ICC/IF on human samples by our Abpromise. Details are at the following link: https://www.abcam.com/index.html?pageconfig=abpromise There are a number of different causes of autofluorescence and various strategies for coping with them. One resource that I have found to be useful is an online guide to autofluorescence produced by the Toronto Western Research Insitute University Health Network. The PDF file can be found at this link: http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf I do not have acces to the following journal, but judging from the title, this paper appears to directly address macrophage autofluorescence: Autofluorescence of alveolar macrophages: problems and potential solutions. Edelson JD, MacFadden DK, Klein M, Rebuck AS. Med Hypotheses. 1985 Aug;17(4):403-7. PMID:3900654 I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Answered on Oct 25 2011