[Histonet] MMP2
Hi Luis, I had good success with mouse monoclonal clone A-Gel2 VC2 from Neomarkers after HIER in citrate buffer. Please, check my paper: Lhotak S. et al, Clin Exp Metastasi 2000, 18(6): 463-70, for conditions, and for staining for other MMPs and TIMPS. It is a bit dated, though, there may be other antibodies available now. I don't work on MMPs anymore. Sarka Lhotak McMaster University, Hamilton, Ontario, Canada ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Annexin V IHC in mouse xenograft
Hi Aprill, I am not sure if you are using an antibody against Annexin V or if you are trying to stain with Annexin V (use it as a detection agent). Annexin V is a protein that binds to phosphatidyl serine. It is often used as a tool to detect early stages of apoptosis when the plasma membrane flips and exposes phosphatidyl serine on the outside of cells, whereas in normal live cells it would be on the inside. It is used eg. in flow cytometry on unfixed cells. Dead cells or permeabilized cells would be all positive as the reagent (Annexin V) would have access to the inside of cells and react with phosphatidyl serine in the inner leaflet of the membrane. I don't think it could work on tissues in paraffin sections. But somebody may prove me wrong... Sarka Lhotak McMaster University, Hamilton, Ontario ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Bond Refine Red for manual IHC
Hello all, I wonder if anyone is using the Bond Polymer Refine Red Detection kit (cat # DS390, Leica) for manual IHC. I've seen some beautiful staining, the technical specialist at Leica, however, told me it is only for Leica automatic stainers. It is an AP stain. Has anybody tried it? Thanks a lot, Sarka Lhotak McMaster University Hamilton, Ontario, Canada ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] TUNEL
Hi Melissa, I use the Trevigen system with great success on mouse tissue using biotinylated dNTPs, Streptavidin HRP and NovaREd. It should work just as well with fluorescence. Mouse thymus is a great positive control. My protocol is as follows: -Xylenes, ethanols, water. -Proteinase K 30 minutes at 37C -Endog. peroxidase quenching: 45 ml methanol+5ml 30% H2O2, 5 min. -1x labeling buffer, 5 min -Reaction mixture: 1x labeling b. 50ul + TdTdNTP mix 1ul+ 50xCo2+ 1ul + TdT enzyme 1ul. 1hr at 37C. -1x Stop buffer, 5 min RT Then I use the Streptavidin HRP and Nova Red that I use for IHC, exactly the same way. I found that the Endog. peroxidase quenching step was ESSENTIAL for this reaction to work, i.e. probably doing more than just quenching, without that there was NO reaction at all. Hope this helps somewhat, don't hesitate to contact me if you have more questions. The beauty of the Trevigen system is that you can buy individual reagents, no need to be paying big bucks for a full kit. (No commercial interest in Trevigen :), just a happy customer). Sarka Lhotak McMaster University, Hamilton ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Mitochodrial IHC marker in FFPE skeletal muscle
Hi all, I need to do a STAT3 IHC on mouse skeletal muscle. It should be showing up in mitochondria. Before purchasing the antibody I am wondering if the mitochodria are well enough preserved in FFPE tissue to show the distinct mitochondrial pattern. Is there a reliable mitochondrial marker (or a good mitochondrial protein antibody) that works on FFPE tissue? Thanks for your help, Sarka Lhotak McMaster university, Hamilton, Ontario ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] image pro plus
Hi Hatem, to separate RGB colours, go to Process/ Colour channel/ Extract. This will give you each channel in BW, i.e. each pixel with intensity from 0 to 255 for Red, Blue and Green component of your image. There must be many ways how to do intensity. One that would be possible is: go to Process/ Pseudocolour/ Select number of divisions (bins). Intensities will then be divided into bins, say 0 to 10, 11 to 20, 21 to 30 etc. all the way to 255. And then look in areas, it will give you number of pixels for each range of intensities you selected in Divisions. Hope it helps, Sarka Lhotak, PhD McMaster University, Hamilton ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
