[Histonet] non-specific fluorescence in artery

[Sarah Tarran]

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_tarran <@t> wmi.usyd.edu.au