Avidin biotin hrp complex

The pancreas from C57BL6 mice was harvested 0, 3 and 7 days post-infection and was frozen in Tissuetek O.T.C. The tissue sections were first fixed in P.A. acetone for 20 min at −20 °C and then incubated with PBS/1% BSA to block non-specific binding. The following incubation included the goat α-CCL17 antibody diluted 1:100 (Santa Cruz Biotechnology, Dallas, TX, USA) in PBS 0.01% saponin overnight. The secondary antibody (rabbit α-goat conjugated to biotin diluted 1:400) was added in PBS and incubated for 2 hours. The sections were then incubated with avidin-biotin-HRP complex (Vector Laboratories, Burlingame, CA, USA) for 30 minutes, and the reaction was revealed with DAB chromogen solution. The tissue sections were washed 3 times in PBS between each incubation step. The tissue was counterstained with H&E, mounted in mounting medium and covered with a coverslip.

Env proteins were detected by SDS-PAGE, BN-PAGE and Western blotting, as previously described [7 (link),25 (link)]. Briefly, Western blotting was performed after transfer of proteins onto a nitrocellulose membrane (Invitrogen, Carlsbad, CA, USA) at 32–40 V for 1–1.5 h. The membranes were blocked with 10% goat serum for 1 h at room temperature and incubated overnight with a 1:2000 dilution of the anti-gp120 MAb ARP 3119 (obtained from the NIBSC Reagent Repository), followed by a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (H + L) and the Western Lightning plus-ECL Enhanced Luminol Reagent (Perkin Elmer, Waltham, MA, USA). Alternatively, the membranes were probed with a HIV-Ig pool derived from individuals infected with viruses from subtypes A, B and C. The subtype A serum was obtained from Stephanie Rainwater (The Fred Hutchinson Cancer Research Center), the subtype C serum from Zdenek Hel (University of Alabama, Birmingham), and the subtype B serum from the AIDS Research and Reference Reagent Program (ARRRP) (product #3957, lot 110180). Each individual serum was present at a 1:3000 dilution in 10 ml of TBS + 0.05% Tween-20. The detection antibody was a 1:3000 dilution of biotin-conjugated goat anti-human IgG, followed by an Avidin-Biotin-HRP complex and a peroxidase substrate kit (all from Vector Laboratories, Burlingame, CA).

Immunohistochemical staining was performed using a standard protocol. Briefly, sections were allowed to dry and were permeabilized for 10 min in 1% Triton X-100 in PBS, followed by blocking in 2% BSA (Roche) and overnight staining at 4°C with the primary antibodies diluted in blocking solution. After endogenous peroxidase inhibition with 3% H₂O₂, sections were incubated with appropriate biotin-conjugate secondary antibody for 1 h at room temperature. Following signal amplification with avidin–biotin-complex–HRP (VECTASTAIN), DAB solution (VECTOR) was applied for 2–3 min. Haematoxylin (Bioptica) was further used to stain nuclei. Haematoxylin and 1% eosin were used for the H&E staining using a standard protocol.
Prussian blue staining was performed using equal parts of hydrochloric acid and potassium ferrocyanide prepared immediately prior to use, which was added to the slides for 20 min. Slides were then washed with distilled water three times and mounted in Mowiol.