Goat anti mouse igm hrp

The levels of secreted total IgM were determined by ELISA. Goat anti-mouse IgM UNLB (SouthernBiotech) and goat anti-mouse IgM HRP (SouthernBiotech) were used as capture and detection Abs, respectively. A 100-μl aliquot of the capture Ab solution diluted with PBS was added to each well of a 96-well microtiter plate and incubated overnight at 4 °C. The plate was washed three times with PBS containing 0.5% Tween 20 (PBS/Tween), and each well was incubated with 200 μl of 4% blockace (Yukijirushi) in H₂O for 1 h at room temperature (RT). The plate was then washed three times with PBS/Tween. A 100-μl aliquot of the supernatant samples diluted with PBS was added to each well and incubated for 2 h at RT. After discarding the supernatants and washing three times with PBS/Tween, 100 μl of a 5×10³ dilution of the detection Ab in PBS was added. After incubation for 1 h at RT, the supernatant was discarded, and the plate was washed three times with PBS/Tween. The enzyme-linked Ab bound to the well was revealed by adding 50 μl/well of 1-Step Ultra TMB solution (Thermo Fisher Scientific). The reaction was terminated by the addition of 2 N sulfuric acid (50 μl/well), and the absorbance at 450 nm was read using a 2030 Multilabel Reader ARVO X3 (PerkinElmer).

Mouse blood samples were taken every 2 weeks. Serum was isolated via centrifugation and stored at −80°C until use. Pneumococcal strains were cultured in THY (THB with 2% yeast extract) and harvested at mid-log phase. The absorbance of the pneumococcal pellet was adjusted to an OD₆₀₀ of 0.1 by dilution with PBS. Then, 96-well immunoplates (SPL) were coated with 100 μl pneumococcal suspension and incubated overnight at 4°C to allow adherence of bacterial cells. The plates were then washed five times with PBS-T, followed by blocking with 1% BSA in PBS for 1 h at RT. After blocking, diluted serum was added to each well and incubated at RT for 1 h, and unbound antibodies were removed by washing with PBS-T. Appropriate dilutions of goat anti-mouse Ig-HRP (Sigma-Aldrich), goat anti-mouse IgG-HRP (Southern-Biotech), or goat anti-mouse IgM-HRP (Southern-Biotech) were added to wells and incubated for 30 min at RT. After washing the plates five times with PBS-T, 100 μl TMB substrate reagent (BD Biosciences, Franklin Lakes, NJ, USA) was added. When colors developed, 50 μl of 2 N H₂SO₄ was added, and the absorbance was measured at 450 nm using a Victor X3 light plate reader (Perkin-Elmer).

For basal serum Ab (IgM/IgG1/IgG2b/IgG2c/IgG3/IgA) measurement, microtiter plates were coated with goat anti-mouse Ig (5 μg/ml, Southern Biotech) overnight at 4 °C. For NP-specific Ab measurement, NP(27)-BSA (Biosearch Technology) or NP(9)-BSA (high affinity) (Biosearch Technology) was used as the capture antigen. Then, nonspecific binding was blocked with 0.5% BSA in PBS for 2 h at 37 °C. Diluted serum samples were incubated in plates for 1 h at 37 °C. Plates were incubated for 1 h with goat anti-mouse IgA-HRP, goat anti-mouse IgM-HRP, goat anti-mouse IgG1-HRP, goat anti-mouse IgG2b-HRP, goat anti-mouse IgG2c-HRP, and goat anti-mouse IgG3-HRP (all from Southern Biotech) and then for 15–30 mins with 100 µl/well TMB (BioLegend) substrate solution, followed by incubation with 50 µL 2 N H2SO4 to stop the reaction. Absorbance values were read at 450 nm using a microplate reader (Cytation5, BioTek).

Three isomers of HAS have been identified and highly conserved among mammalians (HAS1, HAS2, and HAS3), of which HAS2 plays a pivotal role. HAS2 knockout mice resulted in lethal in midgestation due to insufficient development of various organs, whereas other knockouts were not lethal.^{9 (link)} HAS2 also produces larger amount of HA faster than the others,^{24 (link)} which makes HAS2 important for wound repair. Therefore, we detected HAS2 immunoreactivity (ir) in the fascia. We also used hyaluronan-binding protein (HABP) due to its specificity to HA, whereas Alcian blue stains various types of acidic polysaccharides.
The frozen sections were washed with phosphate-buffered saline (PBS), blocked with 10% normal goat serum for 1 hour, incubated with anti-HAS2 (1:200, #sc-365263, Santa Cruz Laboratories, Santa Cruz, CA) or biotinylated HABP (1:250, Merck, Darmstadt, Germany) diluted in 1% normal goat serum incubation buffer overnight at 4°C. After repeated washes with PBS, the samples were incubated with the secondary antibody goat anti-mouse IgM-HRP (1:1000, SouthernBiotech, Birmingham, AL) for 2 hours or HRP-conjugated streptavidin (1:250, Proteintech, Rosemont, IL) for 30 minutes and washed in PBS. The reaction was then developed with 3,30-diaminobenzidine (DAB) and terminated with PBS. Cell quantification is expressed as the number of cells per field.

Goat anti-mouse total IgG_Fc-HRP (Jackson ImmunoResearch), goat anti-mouse IgM-HRP, IgG₁-HRP, IgG_2a-HRP, IgG_2b-HRP, IgG_2c-HRP or IgG₃-HRP (Southern Biotechnology Associates) were used as secondary antibodies (dilutions 1:1000). Antibody was detected using ABTS substrate (Fisher Scientific, NH, USA).

White blood cells (WBC), Red blood cells (RBC), platelets and hematocrit (Hct) were measured using a ScilVet abc plus+ (ScilVet, Oostelbeers, The Netherlands). Plasma triglyceride levels were measured colorimetrically (GPO-PAP, Roche, Woerden, The Netherlands) and plasma total cholesterol was determined enzymatically (CHOD-PAP, Roche, Woerden, The Netherlands) according to the manufacturer's instructions. Total plasma IgM and IgG levels were determined by using a standard ELISA technique. Briefly, plates were coated overnight at 4°C with goat anti-mouse Ig(H+L) (Southern Biotech, Birmingham, LA, USA). Plasma samples were incubated for 2 hours at room temperature followed by a goat-anti-mouse IgM-HRP or goat anti-mouse IgG(H+L) human ads-HRP (Southern Biotech, Birmingham, LA, USA). IgM and IgG levels were visualized by using ABTS Elisa peroxidase substrate (2,5 mg/ml in 0.1M Citrate-phosphate buffer) and measured at 405 nm on a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Specific antibody titers against Cu₂SO₄-oxidized LDL (CuOx-LDL) and malondialdehyde-modified LDL (MDA-LDL) were determined as described previously [20 (link)].