Ab5690

Antibodies for CD3 (Ab5690), CD4 (Ab183685), CD68 (Ab125212), alpha fetoprotein (α-AFP) (Ab46799), and Glypican-3 (GPC-3) (Ab66596) were purchased from Abcam Inc. (Cambridge, MA). Antibodies for SV40 TAg (v-300) (sc-20800) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Antibodies for CD8a (14-0808) and F4/80 (14-4801-81) were purchased from Affymetric eBioscience, (San Diego, CA).

Sections were processed for analysis of cells expressing CD3 (1:50, anti-CD3ε, ab5690, Abcam), Vα7.2 (1:10, 3C10, BioLegend, San Diego, CA, USA), and binding the hMR1 5-OP-RU-PE tetramer (1:50, NIH Tetramer Core Facility, Emory University) [20 (link)]. After deparaffinization and rehydration, sections were treated for antigen retrieval with 10 mM citrate buffer (pH 6.0) and heated (90 °C) for 40 min. Slides were cooled for 30 min in antigen retrieval buffers at room temperature, then washed three times with PBS for 10 min. Sections were incubated overnight with the primary antibody and/or 5-OP-RU tetramer and then for 1 h with fluorescent-dye-conjugated secondary antibodies. All images were acquired and analyzed with an inverted TCS SP5 Leica laser-scanning confocal microscope, using a 40x magnification objective (Leica Microsystems), as previously described [21 (link)].

At one month after pilocarpine induced SE, 6 pilocarpine-treated and 4 control mice were i.p. treated with 75 μl of a 15% xylazine (Rompun®, Bayer) and 85% ketamine (Ketanest® S, Pfizer) mix prior to vascular perfusion. Then, animals were decapitated and whole brains were prepared and incubated with 3.7% paraformaldehyde (commercial 37% PFA solution, Roth CP10.3, Karlsruhe, Germany; diluted to the final concentration in phosphate-buffered saline, pH adjusted to 7.4) for at least 24 hours before they were transferred into a 30% sucrose solution. Cryostat sections (10 μm) were treated for 5 minutes using 10% methanol and 7% H₂O₂ in Tris buffer followed by 1 hour of blocking (10% bovine serum albumin, 1.5% DL-lysine and ~50 μl Triton X-100 in Tris buffer for 4 sections). Incubation with an anti-CD3 antibody (abcam ab5690) was performed over night at room temperature followed by an Alexa-488-conjugated secondary antibody (Invitrogen A11034) for 90 minutes, again at room temperature. Carrier solution for the antibodies was Tris buffer with 2% normal sheep serum. The sections were covered with ProLong® Gold antifade reagent with DAPI (Invitrogen) and evaluated using the Leica DMI6000 B microscope and LAS AF software.

Immunofluorescence was performed on frozen 7 μm OCT- embedded aneurysmal tissue sections. For T cell visualization, rabbit polyclonal anti-human CD3 (ab5690; Abcam, Cambridge, UK) was employed and for macrophages, mouse monoclonal anti-human CD68 (ab955; Abcam, Cambridge, UK) was used. Appropriate secondary antibodies were employed (Donkey anti-rabbit IgG-Alexa Fluor 594 and Donkey anti-mouse IgG—Alexa Fluor 647, ThermoFisher Scientific). Sections treated with secondary antibodies alone did not show specific staining. Staining was visualized on a Zeiss Cell Observer SD confocal microscope (Zeiss, Oberkochen, Germany).