Mca771ga

Floating serial sections were washed in PBS three times for at least 5min at RT. Sections were blocked with 5% normal goat serum (Invitrogen) or normal donkey serum (Sigma) and 0.02% Triton X-100 (Sigma) in PBS for at least 1h at RT. Sections were incubated with primary antibody diluted in blocking solution overnight at 4°C. The primary antibodies used were goat anti-doublecortin (DCX) (Santa Cruz, SC-8066; 1:200; Antibody Registry ID AB_2088494), mouse anti-NeuN (Millipore, MAB377; 1:500; Antibody Registry ID AB_10048713), rat anti-CD11b (Millipore, CBL1313; 1:500; Antibody Registry ID AB_92930), rat anti-CD16/32 (BD Pharmingen, 553141 Clone 2.4G2; 1:800); rabbit anti-CD206 (Abcam, AB64693; 1:50), rabbit anti-GFAP (Dako, Z0334; 1:500; Antibody Registry ID AB_2314535), rabbit anti-Olig2 (Millipore/Chemicon, AB9610; 1:500), rat anti-Ly-6B.2 (Serotec, MCA771GA; 1:1000; Antibody Registry ID AB_324243). Sections were then washed in PBS and incubated with Alexafluor⁴⁸⁸-conjugated donkey anti-goat, goat anti-mouse, goat anti-rabbit or goat anti-rat IgG (all Molecular Probes; 1:500) diluted in PBS, for 2h at RT. Sections were given a final wash with DAPI (1:5000, Invitrogen D1306) in PBS, slide mounted and coverslipped using Mowiol [49 (link)] or Dako fluorescence mounting medium (Dako, Sydney, Australia).

Cryo-embedded brain sections from mice 24 h after induction of stroke were cut into slices (10 µm thick) and fixed in 5% PFA (Sigma-Aldrich, Hamburg, Germany) in PBS (Sigma-Aldrich, Hamburg, Germany) for the staining of neutrophilic granulocytes. To prevent unspecific binding, the slices were pre-treated with 5% BSA in PBS for 45 min. Invading immune cells were detected by rat anti-mouse Ly-6B.2 alloantigen (neutrophilic granulocytes; MCA771GA, Bio Rad, Hercules, CA, USA; 1:500 in PBS supplemented with 1% BSA, overnight at 4 °C). Then, slides were treated with biotinylated anti-rat IgG (BA-4001, Vector Laboratories, Burlingame, CA, USA; 1:100 in PBS supplemented with 1% BSA; 45 min at room temperature). According to the manufacturer’s instructions (Vectorstain ABC Kit, Peroxidase Standard PK-4000, Vector Laboratories, Burlingame, CA, USA), the coupling to biotinylated peroxidase (POD) was performed. Visualization was performed using chromogen 3,3′-diaminobenzidine (DAB; Kem-En-Tec Diagnostics, Taastrup, Denmark). Neutrophilic granulocytes were then counted at the level of the basal ganglia (0.5 mm anterior from bregma) of four different animals using a Nikon microscope Eclipse 50i. This microscope was equipped with the DS-U3 DS camera control unit and the NIS-Elements software (Nikon, Tokio, Japan) [52 (link)].

All histologic analysis were performed as described before [15 (link)]. In brief, lungs were fixed by instillation of a buffered 4% formaldehyde solution under a constant hydrostatic pressure (30 cm H₂O for 15 minutes), kept in 4% formaldehyde solution for additional 24 hours without hydrostatic pressure, embedded in 1% agarose, cut into slices of exactly the same thickness (5 mm), and embedded in paraffin. Primary antibodies for CD3 (rabbit, 1/100, ab5690; Abcam, Cambridge, UK) and Ly6B (rat, 1/150, MCA771GA; Bio-Rad, Munich, Germany) were used for immunohistochemistry analysis blinded to the investigator. Corresponding HRP-conjugated secondary antibodies (anti-rabbit 414341F and anti-rat 414311F, Histofine Simple Stain, Nichirei Biosciences Inc. Japan) were used. Randomly selected fields were evaluated for positive cells using the Visiopharm Integrator System (Visiopharm, Hoersholm, Denmark) on an Olympus BX51 microscope. Paraffin sections were stained with hematoxylin-eosin (H&E), blinded to the investigator and the inflammatory score was calculated as described before [6 (link), 16 (link)]. The mean chord length (MCL) was calculated blinded to the investigator using the Visiopharm Integrator System on an Olympus BX51 microscope equipped with an 8-position slide loader.

The paraffin-fixed kidney sections were deparaffinized, rehydrated, and antigen-retrieved in sodium citrate buffer (10 mM, pH 6.0) for 20 min. The kidney sections were blocked in 10% normal horse serum and incubated with a primary antibody for Ly-6B.2 (MCA771GA; Bio-Rad, Hercules, CA, USA) overnight at 4 °C. The free-floating brain sections were incubated with primary antibody for GFAP (Z033429; Agilent Dako, Santa Clara, CA, USA) overnight at 4 °C. After washing of unbound antibodies, the sections were incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Then, the sections were incubated in avidin-biotin-peroxidase complex solution (ABC solution; Vector Laboratories) for 30 min and developed by using a 3,3’-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA, USA). Finally, the sections were counterstained with hematoxylin and analyzed using a CKX41 light microscope (Olympus, Tokyo, Japan).