Re: [Histonet] IF staining on peritoneal macrophages
Hi Andrea, Jackson lists a couple of FITC-conjugated donkey anti-rat secondaries: 712-095-150http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=PRODProduct_Code=712-095-150 - Whole Donkey Anti-Rat IgG (H+L). 712-096-150http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=PRODProduct_Code=712-096-150 - F(ab')2 fragment Donkey Anti-Rat IgG (H+L). Do you recognize which is the one you have ben using. I have been using Serotec's STAR80F antibody. They suggest using 1:10 Normal Mouse Serum in PBS to make the necessary dilutions. Is this something you do with the Jackson antibodies as well? I've never heard of this technique before. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IF staining on peritoneal macrophages
Hello Histoneters, I am very happy to say that my cytospins are finally working and that cells are staying on the slides throughout the procedure. Thank you very much to all who chimed in or helped with trial samples. I was almost ready to declare victory when I noticed one of my negative control slides looked a bit much like it has specific staining. The antibodies I have been using have not been giving the most even results. Does anyone have a recommendation for an anti-mouse F4/80 antibody? And a matching FITC-conjugated secondary antibody? Is anyone here doing IF staining of mouse peritoneal macrophages? ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IF staining on peritoneal macrophages
Thank you Adam and Jay for your replies. Cytoslides are the pre-marked slides for use with the Cytospin - http://www.thermo.com/com/cda/product/detail/0,1055,21035,00.html Adam, do you have a recommended concentration of BSA for this? Also, do you air dry the slides prior to fixation? Do you air dry afterwards? Thanks again for your help. Mauricio ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IF staining on peritoneal macrophages
Greetings all, I am trying to do immunofluorescence on peritoneal macrophages. I am having a couple of issues that I was hoping one of you could help me resolve. Firstly, I am having uneven results with the Cytospin. Cells tend to get washed off the slides during rinses. Does anyone have tips on how to make cells stick a little better to Cytoslides? Secondly, I have not yet found a satisfactory method for fixation. In order to prevent loss of cells when dipping the slides into fixatives, I am having to air dry the slides before I can do anything to them. I tried the Shandon Collection Fluid (ethanol, isopropanol, carbowax) with great success, but it killed the fluorescence. I am leaning towards spinning the lavage fluid until I get a pellet and then resuspending in PBS with a little BSA added. I hope this makes cells stick well enough that I can put the slides in formalin or acetone. Any thoughts are appreciated! ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
