Goat anti mouse igg h l

Immunohistochemistry was performed on 10 µm-thick pectoralis major muscle cross-sections obtained from the FG-C, FG-WSWB and SG groups. All primary antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH, maintained at The University of Iowa, Department of Biology, Iowa City, IA, and developed by Douglas M. Fambrough, as follows: 39 (anti-collagen VI), B3/D6 (anti-fibronectin), and 31/31-2 (anti-laminin). Secondary antibodies and conjugated streptavidin were obtained from Southern Biotech, Birmingham, AL, USA, as follows: goat anti-mouse IgG1 conjugated to a TR antibody, goat anti-mouse IgG H+L conjugated to a biotin antibody, and streptavidin conjugated to Cy2. The washing procedure consisted of incubating cross-sections for 3 × 5 minutes in phosphate-buffered saline solution. Slides were mounted in Mowiol medium (Sigma, Lyon, France) and stored at 4 °C in a dark chamber before they were observed. Whatever the labelling procedure, six images per sample were selected randomly (captured at 10× and/or 20× magnification using a Leica MC170 camera; Leica Microsystems, Nanterre, France) on a Leica DMRB microscope.

To determine titers of donor-specific antibodies in the serum of recipients, 1 × 10⁶ BALB/c or 2W.OVA.B6 splenocytes were incubated for 30 minutes at 4°C with 5 μL of serum from recipient mice. Cells were then washed and incubated with anti-CD19 (1D3, catalog 550992, BD Biosciences) and goat anti-mouse IgG (H+L) (catalog 1031-02, Southern Biotech) for 30 minutes at 4°C. MFI of the CD19^– cells that were IgG positive was measured by flow cytometry on the Cytek Aurora.