Fluorescein isothiocyanate conjugated anti rabbit igg

The rats were deeply anesthetized with pentobarbital (60 mg/kg, i.p.) on day 14 after the start of PTX (4 mg/kg) treatment, and spinal cords were prepared for immunohistochemistry. Rats were perfused transcardially with 20 mL of potassium-free phosphate-buffered saline (K⁺-free PBS; pH 7.4) followed by 50 mL 4% paraformaldehyde solution. The spinal cord (L_4–6) was removed, post-fixed for 3 h, and cryoprotected overnight in 25% sucrose solution. The spinal cord was stored at −80 °C until use. The spinal cord sections were cut at 10 μm thickness, thaw-mounted on silane-coated glass slides, and air-dried overnight at room temperature. Spinal cord sections were incubated with excess blocking buffer containing 2% skim milk in 0.1% Triton X-100 in K⁺-free PBS, then subsequently reacted overnight at 4 °C with anti-TRPV1 antibody (Alomone Labs, 1:200) in 2% bovine serum albumin/0.1% Triton X-100 in K⁺-free PBS. The sections were then incubated in fluorescein isothiocyanate-conjugated anti-rabbit IgG (Sigma-Aldrich, 1:200) for 2 h at room temperature. All sections were treated with Permafluor (Thermo Shandon, Pittsburgh, PA, USA), cover-slipped, and evaluated with microscopy.

All the serum and aqueous humor samples were tested, by indirect fluorescence antibody test (IFAT), for the presence of the antibodies against L. infantum, T. gondii, A. phagocytophilum and B. caballi using different commercial agent specific slides (Fluoleish, Biovetotest Diagnostic Veterinaire, France, Fuller Laboratories Fullerton, California, USA, MegaFLUO^® ANAPLASMA ph. Horbranz, Austria and Agrolabo, Scarmagno, Italy, for L. infantum, T. gondii, A. phagocytophilum and B. caballi, respectively). For all IFATs, a fluorescein isothiocyanate conjugated anti-rabbit IgG (Sigma-Aldrich, St Luis, MO, USA) was used.
For the detection of antibodies, in both serum and aqueous humor, against L. infantum, T. gondii and A. phagocytophilum, a dilution of 1:25 was used as cut off value, while the threshold value for the detection of antibodies against Babesia spp. was 1:50, as previously reported [22 (link),51 (link),52 (link),53 (link)]. Moreover, a cut off value of 1:10 was used in aqueous humor, for the detection of antibodies against all the above-mentioned microorganisms. A Nikon Eclipse E-400 fluorescence microscope was used for the observation (objective × 100).