Hi Histonetters, We have a problem. We have optimised LSP-1 (leukocyte specific protein) on spleen that has been fixed in 4% PFA/PBS for 2h, incubated in 18%sucrose/PBS overnight and then embedded in OCT. We now want to test it on our arteries that have been fixed and frozen the same way as the spleen. The staining protocol used is also identical for both tissues (see below). However, unlike the specific staining we observe in spleen (on the leukocytes), we observe very obvious non-specific background in the arteries. Pretty much all of the stroma surrounding the tissue is positively staining. The negative controls look good. Could anyone please offer any reasons for what we're seeing? Our method is:
H2O to get rid of OCT Citrate buffer - 10min at 60C (H2O bath) Wash in running water - 10 min Soaked 0.3%Triton/PBS - 3changes, 3 min each Dako Protein block - 10 min Primary Ab (rabbit anti-mouse), 1:50 diln - 1h TBS with tween - 3x 3 changes secondary Ab (Alexa Fluor 594, goat anti-rabbit), 1:800 - 1h TBS with tween - 3x 3 changes DAPI Thanks, Sarah Sarah Tarran Postdoctoral Fellow Vascular Biology Research Centre, Department of Surgery Westmead Hospital, Westmead, NSW, 2145 sarah_tar...@wmi.usyd.edu.au _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet