Incomplete freund adjuvant

Mouse OPN-FL, OPN-N, and OPN-C were cross-linked to ovalbumin (OVA) (Sigma) with glutaraldehyde (Sigma) as reported (26 (link)).
The experimental protocol and animal handling were approved by the ethical committee of the University of Piemonte Orientale (reference no. 10821, 10/2013), Novara, Italy. Four-week-old female C57BL/6 mice (n = 8/10 each group) were anesthetized with isoflurane and immunized weekly intra-peritoneally for 4 weeks with 10 μg of OPN-FL/OVA, 5 μg of OPN-N/OVA or OPN-C/OVA or 10 μg of OVA in 50 μl glycine buffer, 0.15 M, pH 5.7, and 50 μl of incomplete Freund adjuvant (Sigma). EAE was induced 1 week after the last immunization with 200 μg of MOG_35–55 peptide (Espikem, Florence, Italy) and scored as reported (22 (link)).
Mouse splenocytes were purified and cultured in the presence or absence of 10 μg/ml MOG_35–55 as reported. After 5 days, levels of IFN-γ, IL-10, IL-4, and IL-17 were evaluated by ELISA (Biolegend, San Diego, CA, USA) in the supernatants and cell proliferation by incorporation of [³H] thymidine (27 (link)).

Female Dark-Agouti rats (8–10 weeks of age) were purchased from Janvier (France). Five animals were housed in one cage, and they had ad-libitum access to standard chow and water. Animals were acclimated to the environment for at least 1 week before commencement of experiments. Arthritis was induced on day 0 by a single injection of 300 μg bovine type II collagen at the base of the tail (mdbioproducts, Egg bei Zurich, Switzerland) emulsified in an equal volume of incomplete Freund adjuvant (Sigma Aldrich, Deisenhofen, Germany) intradermally at the base of the tail as described previously^{25 (link)}. Rats were inspected for clinical symptoms and weight on a daily basis. The Government of the Oberpfalz approved all experiments according to institutional and governmental regulations for animal use (Az. 54-2532.1-25/13, Az. 55.2-2532-2-413).

All experiments were performed using 8-week-old female Sprague Dawley rats (body weight 180~200g, purchased from Beijing Vital River Laboratory Animal Technology). A total of 140 rats were purchased, maintained and used according to institutional guidelines. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Inner Mongolia Medical University. Animals were housed 2 per cage and kept in the institutional animal facility at a constant temperature and humidity. Food pellets and water were provided ad libitum.
Rheumatoid arthritis was generated by type II collagen (Sigma) immunization as described previously (14). Briefly, type II collagen was dissolved in 0.01mol/L acetic acid overnight at 4°C. Collagen was then emulsified by complete Freund adjuvant (Sigma) and intradermally injected at the tail tip (0.2ml, 1mg/ml) for primary immunization. Two weeks later, type II collagen solution emulsified by incomplete Freund adjuvant (Sigma) was intradermally injected to the inguinal region at the same dose. A total of 100 rats were immunized to generate rheumatoid arthritis model.
Osteoarthritis was established by intra-knee joint cavity injection of L-cysteine papain (20 µl) in 20 rats [15 (link)]. 20 control animals received two intradermal injections of 0.9% saline.

We randomly divided the mice into three groups (n = 27/group). The first group comprised non-vaccinated non-infected mice (negative control). The mice in the second group were orally infected using gastric tubes with 1 × 10⁵C. parvum oocysts in 250 μL PBS (pH = 7.2) [38 (link)] 1 h before a meal on week 4 of the experiment (positive control). The mice in the third group were immunized with the Cryptosporidium-specific purified fraction by two doses of subcutaneous injection at 2-week intervals. The vaccine comprised 40 μg/kg of purified fraction mixed with 250 μL PBS (pH = 7.2) and was emulsified with an equivalent volume of complete Freund adjuvant (Sigma-Aldrich) in the first dose and incomplete Freund adjuvant (Sigma-Aldrich) in the second dose [28 (link)]. Then, the vaccinated mice were orally challenged with C. parvum oocysts after 2 weeks, and the positive control group was infected at the same time. Three mice per group were sacrificed each week, and blood samples were collected from all groups before immunization (day 0) until the 7^th week post-first vaccination dose. The sera were separated and frozen at −20°C for further use. From day 3 post-infection (PI), we collected mice fecal pellets every day for 3 weeks to monitor the oocyst count throughout the experiment. On day 10 PI, we sacrificed three mice from each group to examine histopathological changes in tissues.

All animal experiments were approved by and conducted according to institutional and governmental regulations for experimental animal usage (Ethical Review Committee, Government of the Oberpfalz Az. 54–2532.1-25/13). For arthritis induction, rats were anaesthetized and 300 μg dissolved bovine collagen II (#804001-sol; MD Bioproducts, Egg, Switzerland) emulsified in an equal volume of incomplete Freund adjuvant (Sigma-Aldrich, Taufkirchen, Germany) was intracutaneously injected at the base of the tail. Controls received equal volume of 0.9 % sodium chloride (NaCl) solution (Braun Melsungen AG, Melsungen, Germany). Development of arthritis was monitored by determination of body weight and arthritis score at the respective sampling days. For scoring, each paw was evaluated separately. Three regions were analyzed and one point assigned for signs of inflammation (redness, swelling) occurring in toes/fingers, metatarsus/metacarpus, or ankle. An additional point was given if normal use of the hind limbs was impaired. For front paws, only fingers and metacarpus were evaluated. Altogether, a maximum of 12 points per animal could be assigned (adapted from [9 (link)]).