Infinite m200 spectrofluorimeter

We used serial plasma dilutions (10%, 5%, 2.5%, 1.25%, and 0.75% in BBS) and pooled plasma of mice belonging to the same experimental group, incubating the samples for 15 min on mannan- (10 µg/mL) or acBSA-coated (25 µg/mL) plates prepared according to a procedure reported previously.^{18 (link)} Plates were washed and incubated for 1 h 30 min at RT with a polyclonal anti-human-C3c antibody (Dako, A0062) diluted to 2.4 µg/mL in wash buffer. After washing, plates were incubated with an alkaline phosphatase-labeled goat anti-rabbit IgG antibody (Sigma A-3812) diluted to 1 µg/mL in wash buffer for 1 h 30 min at RT. After washing, the assay was developed by adding 100 µL of substrate solution (Sigma Fast p-Nitrophenyl Phosphate Tablets, Sigma). The absorbance at 405 nm was then measured using the Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).

MBL isoforms were analyzed after sample incubation on mannan-coated (10 µg/mL) plates prepared according to a procedure reported previously.^{18 (link)} Plates were incubated for 1 h at 37 °C with EDTA-plasma diluted in barbital-buffered saline (BBS; 4 mm barbital, 145 mm NaCl, 2 mm CaCl₂, 1 mm MgCl₂ (pH 7.4)) to a final plasma concentration of 2.5% or with cortical homogenates (ipsilateral and contralateral to the lesion) diluted in BBS to a final concentration of 4 mg/mL total protein. Samples were then incubated for 1 h 30 min at room temperature (RT) with anti-MBL-A or anti-MBL-C antibodies (both 1 µg/mL, Hycult, # HM1035 and #HM1038, respectively). After washing, plates were incubated with an HRP-labeled goat anti-mouse IgG antibody (Santa Cruz, CA, USA), diluted to 0.4 µg/mL in wash buffer for 1 h 30 min at RT. After washing, the assay was developed by adding 100 µL of TMB substrate solution (TMB Substrate Kit, code 34021, Thermo Scientific, MA, USA; diluted 1:1 in H₂O₂ solution). The reaction was terminated by adding 100 μL of 2 m H₂SO₄, and the absorbance at 450 nm was measured using an Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).