Mouse anti tnf α

Slide-mounted tissue sections were washed with PBS for 5 min, incubated in
3% hydrogen peroxide for 20 min and washed 3 times in PBS. They were then heated
in 1% antigen-unmasking solution (Vector Laboratories) for 20 min at 90°C,
incubated for 1 h in permeabilization buffer (10% goat serum, 0.1% Triton
X-100 in PBS) and incubated overnight at 4°C with rabbit anti- IL-1β
(1:100) or mouse anti - IL-6 (1:100) or mouse anti-TNFα (1:50) primary antibody
(Abcam) in antibody solution (5% goat serum, 0.05% Triton X-100 in PBS).
Next day, the sections were washed with PBS and incubated 1 h at room temperature with
biotinylated goat anti-rabbit, (1:400), or biolinylated goat anti- mouse (1:400)
antibodies (Vector Laboratories) in antibody solution. Sections were then washed in PBS,
incubated in avidin-biotin complex mixture (ABC,1:100) for 1 h at room temperature, washed
again and developed with diaminobenzidine solution (DAB), washed with PBS, dried and cover
slipped with vectamount mounting medium. For immunostaining of RFP sections were
pre-treated in the same way and incubated with rabbit anti-DsRed (1:1000) primary antibody
(Abcam) over night at 4°C, washed with PBS and incubated with biotinylated goat
anti-rabbit antibody, 1:400, (Vector Laboratories) for 1 h at room temperature. Sections
were washed with PBS, dried and cover slipped with Vectashield aqueous mounting medium
with DAPI.