Fitc conjugated streptavidin

After the behavioral measurement, rats were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and were intracardially perfused with 200–250 ml of 0.1M phosphate-buffered saline, pH 7.4, followed by 200–250 ml of 4% paraformaldehyde phosphate buffer, pH 7.4. The brains were then postfixed at 4°C for 24 h and dehydrated in 30% sucrose for at least 4 days. Serial coronal 30 μm brain sections that contained the medial prefrontal cortex were cut on a Leica freezing microtome and stored in a cryoprotectant solution at -20°C. The sections were incubated overnight at 4°C in a solution of biotin-conjugated lectin wisteria floribunda (WFA, Sigma Aldrich, L1516). All of the sections were then washed 3 times in PBS and then incubated in FITC-conjugated streptavidin (Sigma-Aldrich, S3762) in 25°C for 3 h. Four or five sections from each brain region of each rat were selected. Fluorescence microscope with an image-analysis program was used for measuring the number of WFA-positive PNNs. The average number of PNNs on either side of target brain region was taken as the positive immunoreactive cell number for each rat as previously reported [31 (link),32 (link)].

Exposed haemocytes were fixed as described above, washed in PBS, permeabilised in PBST, and incubated for 30 min to 1% Evans Blue (to quench autofluorescence). Cells were then incubated for 30 min in 10% goat serum in PBS (to reduce nonspecific staining) and with Ab1, Ab2, or Ab3, at the concentration of 10 µg mL⁻¹, for 1 h. After washing in PBS, haemocytes were incubated for 1 h in biotin-conjugated goat anti-mouse IgG antibody (Calbiochem, San Diego, CA, USA), 10 µg mL⁻¹ in PBS, washed again, and exposed to FITC-conjugated streptavidin (Sigma Aldrich, St. Louis, MO, USA). After a final washing, slides were mounted with FluorSave^TM Reagent (Calbiochem, San Diego, CA, USA) and observed under an Olympus (Tokyo, Japan) CX31 light microscope (LM) equipped with an LED fluorescence module with an exciting wavelength of 450 nm (Amplified by Fluorescence Excitation of Radiation Transmitted, AFTER, Fraen Corp. s.r.l., Milan, Italy).

Endotoxin-free RPMI 1640 and miscellaneous culture reagents were purchased from StemCell Technologies (Vancouver, BC, Canada). Fetal calf serum (FCS) was purchased from Gibco Laboratories (Burlington, ON, Canada). Protease inhibitor mixture and PMSF were purchased from Sigma-Aldrich (St. Louis, MO). Sulfo-NHS SS biotin, FITC-conjugated streptavidin and NDSB256 were purchased from Sigma-Aldrich. Phycoerythrin (PE)-conjugated I-A^b-OVA_323-339 tetramer and PE-conjugated I-A^b-ESAT6_1-20 tetramer were purchased from MBL International (Woburn, MA). Phycoerythrin (PE)- conjugated I-A^b-Ag85B_280-294 tetramer was acquired from NIH. 7-AAD was purchased from BD Pharmingen.